Clinical Oral Investigations

, Volume 21, Issue 3, pp 879–888 | Cite as

Ultrasonic activation of irrigants increases growth factor release from human dentine

  • M. WidbillerEmail author
  • A. Eidt
  • K.-A. Hiller
  • W. Buchalla
  • G. Schmalz
  • K. M. Galler
Original Article



Bioactive proteins are sequestered in human dentine and play a decisive role in dental pulp regeneration and repair. They can be released and exposed on the dentine surface by acids, but also chelators, such as ethylenediaminetetraacetic acid (EDTA). The objectives of this study were (i) to evaluate whether ultrasonic activation of irrigants in the root canal will promote growth factor release from dentine and (ii) to collect bioactive proteins in a physiological solution.

Materials and methods

Human dentine disks underwent irrigation with and without ultrasonic activation. The protocols included treatment by either a single or two consecutive steps with 10 % EDTA and phosphate-buffered saline (PBS), where each sample was treated three times. To mimic clinical conditions, selected irrigation regimens were applied to root canals of extracted human teeth after preparation. Amounts of transforming growth factor β1 (TGF-β1) in solution were quantified using enzyme-linked immunosorbent assays. Nonparametric statistical analysis was performed to compare different groups as well as repetitions within a group (Mann-Whitney U test, α = 0.05). Additionally, morphological changes of dentine surfaces were visualized by scanning electron microscopy (SEM).


TGF-β1 was not detectable after irrigation of dentine with PBS, neither with nor without ultrasonic activation. Irrigation with EDTA released TGF-β1, and ultrasonic activation of EDTA enhanced this effect. However, preceding EDTA conditioning enabled the release of bioactive proteins into PBS solution. Similar results were observed in dentine disks and root canals. Visualization of dentine surfaces after different treatment revealed superficial erosion after ultrasonic activation irrespective of the irrigant solution, but different degrees of exposure of organic substance.


Ultrasonic activation enhances growth factor release from human dentine. Bioactive proteins can be isolated in physiological solvents and may act as autologous supplements for regenerative endodontic treatment or pulp tissue engineering.

Clinical relevance

Autologous growth factors from human dentine can advance treatment strategies in dental pulp tissue engineering.


Ultrasonic activation Dentine Transforming growth factor beta1 Ethylenediaminetetraacetic acid Tissue engineering Regenerative endodontic procedure 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


The work was supported by the Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Germany.

Informed consent

Human tissue was obtained according to an informed consent protocol approved by an appropriate review board at the University of Regensburg.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. 1.
    Smith AJ, Scheven BA, Takahashi Y, et al. (2012) Dentine as a bioactive extracellular matrix. Arch Oral Biol 57:109–121CrossRefPubMedGoogle Scholar
  2. 2.
    Goldberg M, Smith AJ (2004) Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med 15:13–27CrossRefPubMedGoogle Scholar
  3. 3.
    Dung SZ, Gregory RL, Li Y, Stookey GK (1995) Effect of lactic acid and proteolytic enzymes on the release of organic matrix components from human root dentin. Caries Res 29:483–489CrossRefPubMedGoogle Scholar
  4. 4.
    Tomson PL, Grover LM, Lumley PJ, et al. (2007) Dissolution of bio-active dentine matrix components by mineral trioxide aggregate. J Dent 35:636–642CrossRefPubMedGoogle Scholar
  5. 5.
    Graham LW, Cooper PR, Cassidy N, et al. (2006) The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components. Biomaterials 27:2865–2873CrossRefPubMedGoogle Scholar
  6. 6.
    Ferracane JL, Cooper PR, Smith AJ (2013) Dentin matrix component solubilization by solutions of pH relevant to self-etching dental adhesives. J Adhes Dent 15:407–412PubMedGoogle Scholar
  7. 7.
    Galler KM, Widbiller M, Buchalla W, et al. (2015) EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells. Int Endod J. doi: 10.1111/iej.12492
  8. 8.
    Zehnder M (2006) Root canal irrigants. J Endod 32:389–398CrossRefPubMedGoogle Scholar
  9. 9.
    Chun SY, Lee HJ, Choi YA, et al. (2011) Analysis of the soluble human tooth proteome and its ability to induce dentin/tooth regeneration. Tissue Eng Part A 17:181–191CrossRefPubMedGoogle Scholar
  10. 10.
    Baker SM, Sugars RV, Wendel M, et al. (2009) TGF-beta/extracellular matrix interactions in dentin matrix: a role in regulating sequestration and protection of bioactivity. Calcif Tissue Int 85:66–74CrossRefPubMedGoogle Scholar
  11. 11.
    Finkelman RD, Mohan S, Jennings JC, et al. (1990) Quantitation of growth factors IGF-I, SGF/IGF-II, and TGF-beta in human dentin. J Bone Miner Res 5:717–723CrossRefPubMedGoogle Scholar
  12. 12.
    Roberts-Clark DJ, Smith AJ (2000) Angiogenic growth factors in human dentine matrix. Arch Oral Biol 45:1013–1016CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang R, Smith AJ, Cooper PR, et al. (2011) Angiogenic activity of dentin matrix components. J Endod 37:26–30CrossRefPubMedGoogle Scholar
  14. 14.
    Suzuki T, Lee CH, Chen M, et al. (2011) Induced migration of dental pulp stem cells for in vivo pulp regeneration. J Dent Res 90:1013–1018CrossRefPubMedGoogle Scholar
  15. 15.
    Howard C, Murray PE, Namerow KN (2010) Dental pulp stem cell migration. J Endod 36:1963–1966CrossRefPubMedGoogle Scholar
  16. 16.
    Tziafas D, Alvanou A, Panagiotakopoulos N, et al. (1995) Induction of odontoblast-like cell differentiation in dog dental pulps after in vivo implantation of dentine matrix components. Arch Oral Biol 40:883–893CrossRefPubMedGoogle Scholar
  17. 17.
    He H, Yu J, Liu Y, et al. (2008) Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro. Cell Biol Int 32:827–834CrossRefPubMedGoogle Scholar
  18. 18.
    Melin M, Joffre-Romeas A, Farges JC, et al. (2000) Effects of TGFbeta1 on dental pulp cells in cultured human tooth slices. J Dent Res 79:1689–1696CrossRefPubMedGoogle Scholar
  19. 19.
    Kalyva M, Papadimitriou S, Tziafas D (2010) Transdentinal stimulation of tertiary dentine formation and intratubular mineralization by growth factors. Int Endod J 43:382–392CrossRefPubMedGoogle Scholar
  20. 20.
    Mullane EM, Dong Z, Sedgley CM, et al. (2008) Effects of VEGF and FGF2 on the revascularization of severed human dental pulps. J Dent Res 87:1144–1148CrossRefPubMedGoogle Scholar
  21. 21.
    Galler KM, D'Souza RN, Federlin M, et al. (2011) Dentin conditioning codetermines cell fate in regenerative endodontics. J Endod 37:1536–1541CrossRefPubMedGoogle Scholar
  22. 22.
    Lovelace TW, Henry MA, Hargreaves KM, Diogenes AR (2011) Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 37:133–138CrossRefPubMedGoogle Scholar
  23. 23.
    Chrepa V, Henry MA, Daniel BJ, Diogenes AR (2015) Delivery of apical mesenchymal stem cells into root canals of mature teeth. J Dent Res 94:1653–1659CrossRefPubMedGoogle Scholar
  24. 24.
    Casagrande L, Demarco FF, Zhang Z, et al. (2010) Dentin-derived BMP-2 and odontoblast differentiation. J Dent Res 89:603–608CrossRefPubMedGoogle Scholar
  25. 25.
    Pang NS, Lee SJ, Kim E, et al. (2014) Effect of EDTA on attachment and differentiation of dental pulp stem cells. J Endod 40:811–817CrossRefPubMedGoogle Scholar
  26. 26.
    Carreira AC, Lojudice FH, Halcsik E, et al. (2014) Bone morphogenetic proteins: facts, challenges, and future perspectives. J Dent Res 93:335–345CrossRefPubMedGoogle Scholar
  27. 27.
    Lin LM, Ricucci D, Huang GTJ (2014) Regeneration of the dentine-pulp complex with revitalization/revascularization therapy: challenges and hopes. Int Endod J 47:713–724CrossRefPubMedGoogle Scholar
  28. 28.
    Diogenes AR, Henry MA, Teixeira FB, Hargreaves KM (2013) An update on clinical regenerative endodontics. Endod Top 28:2–23CrossRefGoogle Scholar
  29. 29.
    Becerra P, Ricucci D, Loghin S, et al. (2014) Histologic study of a human immature permanent premolar with chronic apical abscess after revascularization/revitalization. J Endod 40:133–139CrossRefPubMedGoogle Scholar
  30. 30.
    Ballal NV, Kundabala M, Bhat S, et al. (2009) A comparative in vitro evaluation of cytotoxic effects of EDTA and maleic acid: root canal irrigants. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:633–638CrossRefPubMedGoogle Scholar
  31. 31.
    Malheiros CF, Marques MM, Gavini G (2005) In vitro evaluation of the cytotoxic effects of acid solutions used as canal irrigants. J Endod 31:746–748CrossRefPubMedGoogle Scholar
  32. 32.
    Sceiza MF, Daniel RL, Santos EM, Jaeger MM (2001) Cytotoxic effects of 10 % citric acid and EDTA-T used as root canal irrigants: an in vitro analysis. J Endod 27:741–743CrossRefPubMedGoogle Scholar
  33. 33.
    Schmidt TF, Teixeira CS, Felippe MCS, et al. (2015) Effect of ultrasonic activation of irrigants on smear layer removal. J Endod 41:1359–1363CrossRefPubMedGoogle Scholar
  34. 34.
    Mozo S, Llena C, Forner L (2012) Review of ultrasonic irrigation in endodontics: increasing action of irrigating solutions. Med Oral Patol Oral Cir Bucal 17:e512–e516CrossRefPubMedGoogle Scholar
  35. 35.
    Guerisoli DMZ, Marchesan MA, Walmsley AD, et al. (2002) Evaluation of smear layer removal by EDTAC and sodium hypochlorite with ultrasonic agitation. Int Endod J 35:418–421CrossRefPubMedGoogle Scholar
  36. 36.
    Lui JN, Kuah HG, Chen NN (2007) Effect of EDTA with and without surfactants or ultrasonics on removal of smear layer. J Endod 33:472–475CrossRefPubMedGoogle Scholar
  37. 37.
    Castagna F, Rizzon P, da Rosa RA, et al. (2013) Effect of passive ultrassonic instrumentation as a final irrigation protocol on debris and smear layer removal—a SEM analysis. Microsc Res Tech 76:496–502CrossRefPubMedGoogle Scholar
  38. 38.
    Ruggeri A, Prati C, Mazzoni A, et al. (2007) Effects of citric acid and EDTA conditioning on exposed root dentin: an immunohistochemical analysis of collagen and proteoglycans. Arch Oral Biol 52:1–8CrossRefPubMedGoogle Scholar
  39. 39.
    Higashi T, Okamoto H (1995) The effect of ultrasonic irrigation before and after citric acid treatment on collagen fibril exposure: an in vitro SEM study. J Periodontol 66:887–891CrossRefPubMedGoogle Scholar
  40. 40.
    Smith AJ, Duncan HF, Diogenes AR, et al. (2016) Exploiting the bioactive properties of the dentin-pulp complex in regenerative endodontics. J Endod 42:47–56CrossRefPubMedGoogle Scholar
  41. 41.
    Galler KM, Buchalla W, Hiller KA, et al. (2015) Influence of root canal disinfectants on growth factor release from dentin. J Endod 41:363–368CrossRefPubMedGoogle Scholar
  42. 42.
    Galler KM, Eidt A, Schmalz G (2014) Cell-free approaches for dental pulp tissue engineering. J Endod 40:S41–S45CrossRefPubMedGoogle Scholar
  43. 43.
    Diogenes AR, Ruparel NB, Teixeira FB, Hargreaves KM (2014) Translational science in disinfection for regenerative endodontics. J Endod 40:S52–S57CrossRefPubMedGoogle Scholar
  44. 44.
    Althumairy RI, Teixeira FB, Diogenes AR (2014) Effect of dentin conditioning with intracanal medicaments on survival of stem cells of apical papilla. J Endod 40:521–525CrossRefPubMedGoogle Scholar
  45. 45.
    Carrigan PJ, Morse DR, Furst ML, Sinai IH (1984) A scanning electron microscopic evaluation of human dentinal tubules according to age and location. J Endod 10:359–363CrossRefPubMedGoogle Scholar
  46. 46.
    Radlanski RJ (2011) Curriculum Orale Struktur- und Entwicklungsbiologie. Quintessenz, BerlinGoogle Scholar
  47. 47.
    de Gregorio C, Arias A, Navarrete N, et al. (2013) Effect of apical size and taper on volume of irrigant delivered at working length with apical negative pressure at different root curvatures. J Endod 39:119–124CrossRefPubMedGoogle Scholar
  48. 48.
    Tay FR, Gu LS, Schoeffel GJ, et al. (2010) Effect of vapor lock on root canal debridement by using a side-vented needle for positive-pressure irrigant delivery. J Endod 36:745–750CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Lumley PJ, Walmsley AD, Laird WR (1991) Streaming patterns produced around endosonic files. Int Endod J 24:290–297CrossRefPubMedGoogle Scholar
  50. 50.
    Ahmad M, Pitt Ford TJ, Crum LA (1987) Ultrasonic debridement of root canals: acoustic streaming and its possible role. J Endod 13:490–499CrossRefPubMedGoogle Scholar
  51. 51.
    Niu W, Yoshioka T, Kobayashi C, Suda H (2002) A scanning electron microscopic study of dentinal erosion by final irrigation with EDTA and NaOCl solutions. Int Endod J 35:934–939CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • M. Widbiller
    • 1
    Email author
  • A. Eidt
    • 1
  • K.-A. Hiller
    • 1
  • W. Buchalla
    • 1
  • G. Schmalz
    • 1
    • 2
  • K. M. Galler
    • 1
  1. 1.Department of Conservative Dentistry and PeriodontologyUniversity Hospital RegensburgRegensburgGermany
  2. 2.School of Dental MedicineUniversity of BernBernSwitzerland

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