Effects of Reducing Agents on Birefringence Dentin Collagen after Use of Different Endodontic Auxiliary Chemical Substances

Published:August 08, 2011DOI:



      The aim of this study was to evaluate the effect of 10% ascorbic acid or 10% sodium ascorbate on organic matrix collagen of bovine dentin root canal walls after irrigation with 5.25% sodium hypochlorite (NaOCl), 17% ethylenediaminetetraacetic acid (EDTA), or 0.9% sodium chloride.


      Eighty bovine incisors were randomly divided into 8 groups (n = 10): group 1, 0.9% sodium chloride (control); group 2, 5.25% NaOCl + 17% EDTA (NaOCl + EDTA); group 3, 5.25% NaOCl + 17% EDTA + 10% ascorbic acid (NaOCl + EDTA + AA); group 4, 5.25% NaOCl + 17% EDTA + 10% sodium ascorbate (NaOCl + EDTA + SA); group 5, 5.25% NaOCl (NaOCl); group 6, 17% EDTA; group 7, 10% ascorbic acid (AA); and group 8, 10% sodium ascorbate (SA). Teeth were chemomechanically prepared, submitted to histologic processing, and stained with Sirius Red dye to be analyzed under polarized light microscopy. Absorbance assay was also performed to confirm the loss of collagen.


      NaOCl + EDTA and NaOCl groups presented a significantly different birefringence pattern compared with the control group (P < .05). The measurement of the optical retardations of NaOCl + EDTA + SA indicated that this group was not statistically different from the control group. Although the measurement of the optical retardations of NaOCl + EDTA + AA was statistically different from the control group, the results were significantly higher than for NaOCl + EDTA. The birefringence of EDTA, AA, and SA groups was not statistically different from that of control group. The absorbance assay of NaOCl + EDTA and NaOCl groups confirmed the loss of collagen (P < .05).


      It is possible to conclude that 5.25% NaOCl, whether associated or not with 17% EDTA, causes birefringence alterations and loss of dentin collagen. These alterations reduced the ability of Sirius Red to bind with collagen fiber molecules. The reductions in the optical retardation values could be reversed by the application of either 10% ascorbic acid or 10% sodium ascorbate after 5.25% sodium hypochlorite and 17% EDTA irrigation.

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        • Byström A.
        • Sundqwist G.
        Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in the endodontic therapy.
        Scand Dent Res. 1981; 89: 321-328
        • Peters O.A.
        • Laib A.
        • Gohring T.N.
        • et al.
        Changes in root canal geometry after preparation assessed by high resolution computed tomography.
        J Endod. 2001; 27: 1-6
        • Marending M.
        • Paqué F.
        • Fischer J.
        • et al.
        Impact of irrigant sequence on mechanical properties of human root dentin.
        J Endod. 2007; 33: 1325-1328
        • Zehnder M.
        Root canal irrigants.
        J Endod. 2006; 32: 389-398
        • Slutzky-Goldberg I.
        • Maree M.
        • Liberman R.
        • et al.
        Effect of sodium hypochlorite on dentin microhardness.
        J Endod. 2004; 30: 880-882
        • Nikaido T.
        • Takano Y.
        • Sasafuchi Y.
        • et al.
        Bond strengths to endodontically-treated teeth.
        Am J Dent. 1999; 12: 177-180
        • Ishizuka T.
        • Kataoka H.
        • Yoshioka T.
        • et al.
        Effect of NaOCl treatment on bonding to root canal dentin using a new evaluation method.
        Dent Mater J. 2001; 20: 24-33
        • Ari H.
        • Yaşar E.
        • Belli S.
        Effects of NaOCl on bond strengths of resin cements to root canal dentin.
        J Endod. 2003; 29: 248-251
        • Santos J.N.
        • Carrilho M.R.
        • de Goes M.F.
        • et al.
        Effect of chemical irrigants on the bond strength of a self-etching adhesive to pulp chamber dentin.
        J Endod. 2006; 32: 1088-1090
        • Rueggeberg F.A.
        • Margeson D.H.
        The effect of oxygen inhibition on an unfilled/filled composite system.
        J Dent Res. 1990; 69: 1652-1658
        • Morris M.D.
        • Lee K.W.
        • Agee K.A.
        • Bouillaguet S.
        • Pashley D.H.
        Effects of sodium hypochlorite and RC-Prep on bond strengths of resin cement to endodontic surfaces.
        J Endod. 2001; 27: 753-757
        • Nagpal R.
        • Tewari S.
        • Gupta R.
        Effect of various surface treatments on the microleakage and ultrastructure of resin-tooth interface.
        Oper Dent. 2007; 32: 16-23
        • da Cunha L.F.
        • Furuse A.Y.
        • Mondelli R.F.
        • Mondelli J.
        Compromised bond strength after root dentin deproteinization reversed with ascorbic acid.
        J Endod. 2010; 36: 130-134
        • Lai S.C.
        • Mak Y.F.
        • Cheung G.S.
        • et al.
        Reversal of compromised bonding to oxidized etched dentin.
        J Dent Res. 2001; 80: 1919-1924
        • Torabinejad M.
        • Handysides R.
        • Khademi A.
        • Bakland L.
        Clinical implications of the smear layer in endodontics: a review.
        Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002; 94: 658-666
        • Rotstein I.
        • Dankner E.
        • Goldman A.
        • et al.
        Histochemical analysis of dental hard tissues following bleaching.
        J Endod. 1996; 22: 23-26
        • Kishen A.
        • Sum C.P.
        • Mathew S.
        • Lim C.T.
        Influence of irrigation regimens on the adherence of Enterococcus faecalis to root canal dentin.
        J Endod. 2008; 34: 850-854
        • Ballal N.V.
        • Mala K.
        • Bhat K.S.
        Evaluation of the effect of maleic acid and ethylenediaminetetraacetic acid on the microhardness and surface roughness of human root canal dentin.
        J Endod. 2010; 36: 1385-1388
        • Vilarta R.
        • Vidal B.C.
        Anisotropic and biomechanical properties of tendons modified by exercise and denervation: aggregation and macromolecular order in collagen bundles.
        Matrix. 1989; 9: 55-61
        • Constantine V.S.
        • Mowry R.W.
        Selective staining of human dermal collagen: II—the use of Picrosirius Red F3BA with polarization microscopy.
        J Invest Dermatol. 1968; 50: 419-423
        • Piérard G.E.
        Sirius red polarization method is useful to visualize the organization of connective tissues but not the molecular composition of their fibrous polymers.
        Matrix. 1989; 9: 68-71
        • Moreira D.M.
        • Almeida J.F.
        • Ferraz C.C.
        • Gomes B.P.
        • Line S.R.
        • Zaia A.A.
        Structural analysis of bovine root dentin after use of different endodontics auxiliary chemical substances.
        J Endod. 2009; 35: 1023-1027
        • Pearse A.G.E.
        Histochemistry: theoretical and applied.
        3rd ed. Churchill Livingstone, London1972
        • do Espírito Santo A.R.
        • Novaes P.D.
        • Line S.R.P.
        Anisotropic properties of enamel organic extracellular matrix.
        Eur J Oral Sci. 2006; 114: 333-337
        • Prati C.
        • Chersoni S.
        • Pashley D.H.
        Effect of removal of surface collagen fibrils on resin-dentin bonding.
        Dent Mater. 1999; 15: 323-331
        • Celik C.
        • Erkut S.
        • Gulsahi K.
        • Yamanel K.
        • Kucukesmen C.
        Effect of sodium ascorbate on bond strength of different adhesive systems to NaOCl-treated dentin.
        Aust Endod J. 2010; 36: 12-18
        • Hawkins C.L.
        • Davies M.J.
        Hypochlorite-induced oxidation of proteins in plasma: formation of chloramines and nitrogen-centred radicals and their role in protein fragmentation.
        Biochem J. 1999; 340: 539-548
        • Hashimoto M.
        • Tay F.R.
        • Ohno H.
        • et al.
        SEM and TEM analysis of water degradation of human dentinal collagen.
        J Biomed Mater Res B Appl Biomater. 2003; 66: 287-298
        • Pashley D.H.
        • Tay F.R.
        • Yiu C.
        • et al.
        Collagen degradation by host-derived enzymes during aging.
        J Dent Res. 2004; 83: 216-221
        • Carrilho M.R.O.
        • Geraldeli S.
        • Tay F.
        • et al.
        in vivo preservation of the hybrid layer by chlorhexidine.
        J Dent Res. 2007; 86: 529-533
        • Kuboki Y.
        • Okuguchi M.
        • Takita H.
        • et al.
        Amino-terminal location of pyridinoline in dentin collagen.
        Connect Tissue Res. 1993; 29: 99-110
        • Daumer K.M.
        • Khan A.U.
        • Steinbeck M.J.
        Chlorination of pyridinium compounds: possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation.
        J Biol Chem. 2000; 275: 34681-34692
        • Puchtler H.
        • Meloan S.N.
        • Waldrop F.S.
        Are picro-dye reactions for collagens quantitative? chemical and histochemical considerations.
        Histochemistry. 1988; 88: 243-256
        • Junqueira L.C.
        • Bignolas G.
        • Brentani R.R.
        Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.
        Histochem J. 1979; 11: 447-455
        • Carr A.C.
        • Tijerina T.
        • Frei B.
        Vitamin C protects against and reverses specific hypochlorous acid- and chloramine-dependent modifications of low-density lipoprotein.
        Biochem J. 2000; 346: 491-499
        • Weston C.H.
        • Ito S.
        • Wadgaonkar B.
        • Pashley D.H.
        Effects of time and concentration of sodium ascorbate on reversal of NaOCl-induced reduction in bond strengths.
        J Endod. 2007; 33: 879-881
        • VanDuijn M.M.
        • Tijssen K.
        • VanSteveninck J.
        • Van Den Broek P.J.
        • Van Der Zee J.
        Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor.
        J Biol Chem. 2000; 275: 27720-27725
        • Smallwood C.L.
        Drinking water criteria document for chloramines: Health and Ecological Criteria Division, Office of Science and Technology, Office of Water, Technical Support Services.
        Environmental Criteria and Assessment Office, Cincinnati, OH1994