endodontcs ’ Editor. SAMUEL



Department of Endodontology School of Dentistry Temple University 3223 North Broad Street Philadelphia, Pennsylvania 19140

In vitro determination and quantification of 30% hydrogen peroxide penetration through dentin and cementum during bleaching Ilan Rotstein, CD,a Jerusalem, Israel HEBREW








Bleaching pulpless teeth with 30% hydrogen peroxide was reported to cause external cervical root resorption. It was hypothesized that H202 penetrated open dentin tubules to initiate an inflammatory response that could result in root resorption. No direct experimental evidence was provided to support this theory. An in vitro model for determination and quantification of H202 penetration during bleaching procedures is suggested. Artificial defects of the cementum covering the cementoenamel junction were performed in 22 extracted human premolars. After 15 minutes of thermocatalytic bleaching, H202 could be detected in the medium surrounding all the tested teeth. Cervical root permeability to 30% H202 could reach up to 82% of the total amount applied. (ORAL SURC ORAL MED ORAL PATHOL 1991;72:602-6)


t has been demonstrated that intracoronal bleaching of pulpless teeth with 30% hydrogen peroxide may result in cervical root resorption.‘-7 The bleachingrelated cervical resorption may be arrested or progressive, or may result in ankylosis.4, 7 These complications may lead to extraction of the tooth or to rather complicated operative procedures, leaving unesthetic sequela.2$ 5*6, * Several authors postulated that bleaching agents such as Hz02 penetrated through the dentinal tubules Supported by a grant from the Joining Research Fund of the Hebrew University-Hadassah Faculty of Dental Medicine, founded by the Alpha Omega Fraternity, and from the Hadassah Medical Organization. aLecturer, Department of Endodontics. 7/15f 28822 602

to initiate an inflammatory reaction that could cause cervical root resorption. l, 2,4 More recently in vivo studies have demonstrated that internal bleaching with Hz02 caused root resorption in dogs’ teeth.9 Other in vitro studies advocated the capacity of bleaching materials, placed in the pulp chamber of human teeth, to alter the pH of the medium surrounding the root. lo, l1 These studies gave only indirect evidence as to the leakage capacity of such materials and did not relate specifically to the presence of each bleaching agent in the surrounding medium. The purpose of this study was to suggest an in vitro model for direct determination of Hz02 penetration through dentin and cementum during bleaching. In addition, this model permits the quantification of the amount of Hz02 penetrated during the procedure.

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Penetrance of hydrogen peroxide during bleaching


MATERIAL AND METHODS Material preparation

Freshly extracted intact uniradicular premolars, removed for orthodontic reasonsfrom young adults, were used. The teeth with no detectable caries, restorations, or fractures were immediately placed in saline solution, and the soft-tissue debris covering the root surface was gently removed. Care was taken not to damage the radicular cementum. The teeth were subjected to stereomicroscopic examination of the radicular cementum and the cementoenameljunction (CEJ). Only teeth without apparent cementum defects or dentin exposuresat the CEJ were included in this study. Twenty-two teeth were selected for the experiment, of which two served as controls. Root canal treatment was performed in each tooth and included biomechanical preparation followed by an obturation with the lateral condensation technique of gutta-percha and AH26 root canal sealer (De Trey Dentsply, Zurich, Switzerland). The coronal level of the root canal filling was left 3 mm short of the CEJ. The CEJ reference points were either on the buccal or the lingual side. Remnants of gutta-percha and sealer from the pulp chamber and the coronal accesscavity were removed by a 28 mm round carbide bur, rotated at a slow speed, and chloroform. This was followed by thorough rinsing of the pulp chambers with bidistilled water. In each tooth the cementum covering the CEJ was removed in four points-mesial, distal, buccal, and lingual-leaving the dentin tubules in those areas exposed. This was done with a round carbide bur, mounted on a slow-speedhandpiece, to simulate defects or irregularities along the CEJ line. Study


Each tooth was mounted on a 2 X 4 cm laminate of boxing wax (Kerr Brand, Emeryville, Calif.) in such a manner that on one sidethe tooth crown and the accesscavity were exposed,and on the other sidethe root and the CEJ. The wax laminates were adapted at a level of 1 mm above the mesial or distal line of the CEJ. To achieve an hermetic coronal seal, the circumferential union line between the boxing wax and the enamel was sealedwith sticky wax and a double layer of varnish. The outer root surface of the teeth including the apical foramina was sealed with wax, leaving the coronal third and the CEJ exposed. The prepared teeth mounted on the wax laminates were placed in plastic assay tubes containing 1.75 ml bidistilled water with their entire root, including the CEJ, submerged in the solution (Fig. 1). Before the experiment a zero reading of all the sampleswas performed to exclude the possibility of



‘CEJ Gutta -Percha


1. Study model:schematicdiagram.

presenceof Hz02 in the tested system. The teeth were then placed in an incubator for 20 minutes at 37” C to simulate body temperature. Twenty microliters of 30% Hz02 (8.8 mol/L) were pipetted in each access cavity of the 20 experimental teeth. Twenty microliters was used becauseit was found to be the quantity able to saturate a small cotton pellet. In the control teeth 20 ~1 of bidistilled water was used instead of H202. The teeth were subjected to 15 cycles of l-minute thermocatalytic bleaching with a 1000 W heat lamp from a distance of 50 cm. The interval between each heating cycle was of 30 seconds. Measurement

of H202

Aliquots of 0.5 ml from the solution surrounding each root were placed in a test tube containing 0.5 ml of bidistilled water to reach a total volume of 1 ml. Quantification of the Hz02 was done using a specific assay.I2 The Hz02 present in the assay system was added to ferrous ammonium chloride resulting in ferric ion release. Upon the addition of potassium thiocyanate a ferrithiocyanate complex results, which absorbs light at the wavelength of 480 nm. The amount of Hz02 in the samplestested is determined













2. Standardcurve generatedby 30%Hz02 doseresponse. Fig.

by comparing them with a standard curve generated by known amounts of H202. The reaction is colorimetric, and the presence of Hz02 in the system changes the reagents’ color from colorless to red. A standard curve for the 30% Hz02 sourceusedfor the experiment was established (Fig. 2). Samples from each tooth tested were read in a spectrophotometer and compared with the standard curve. Each sample was taken in duplicate and the whole experiment repeated several times. Two additional controls for the H202 and the reagents were taken. The reagents’ readings were a negative control, whereas the reading of pure 20 ~1of 30% Hz02 in 1.75 ml bidistilled water were a positive control. RESULTS

The results are the mean recordings of three repeated experiments and are summarized in Table I. All the tested teeth allowed the penetration of 30% H202, which could be detected in the medium surrounding the roots. The amount of Hz02 penetrated varied from 3 to 83,348 nmol. The permeability of the teeth tested varied from 0.002% to 82.1%. The negative controls were zero nmol, whereas the positive controls were equivalent to 101,500 nmol. DISCUSSION

Since the early reports associating bleaching proceduresof pulplessteeth with external root resorption, severaletiologic mechanismswere proposed.‘, 2*4Harrington and Natkin’ suggested that Hz02 used for bleaching could initiate an inflammatory resorptive processby diffusing through patent dentinal tubules into the cervical periodontal ligament. Lado et al.2 hypothesized that the bleaching agents penetrating patent dentinal tubules may cause dentin denatur-


199 I

1. Results of Hz02 penetration in tested teeth

2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 Control

9 25 14 123 136 83,348 IO 9 49,679 21,491 7 179 3 50 6 117 36 12,615 27 27 0

0.008 0.024 0.013 0.121 0.133 82.116 0.009 0.008 48.944 21.173 0.006 0.176 0.002 0.049 0.005 0.115 0.035 12.428 0.026 0.026 0

ation at the CEJ, where a defect between the enamel and the cementum might be present. This denatured dentin acts as a foreign body and is attacked by elements from the periodontal tissues. Cvek and Lindval14 stated that after the seepageof Hz02 and its initial effect on the periodontium, bacteria may colonize the empty tubules, causing inflammation in the surrounding tissuesaswell asprogressiveroot resorption. The bacteria may originate from the gingival crevices or the pulp chamber. The diffusion capacity of different substancessuch as ions, molecules, solutions, and drugs through dentin and cementum is well documented.13-22This capacity dependson various factors such asthe nature of the penetrating agent, the nature of the dental tissue, the surface area exposedand its location, the remaining dentin thickness, previously applied materials, the presenceof smear layer, and the temperature. Different materials have different penetrating qualities. Smaller mollecules usually diffuse faster than larger ones.13,t4 Hydrogen ions from strong inorganic acids were found to diffuse poorly through dentin, much lessthan hydrogen ions from weak acids and hydroxyl ions from alkaline materials.‘s Fuss et al.” compared the diffusion capacity of a mixture of sodium perborate and 30% Hz02 solution (Super0x01) used asa walking bleach agent, versus calcium hydroxide paste. The tested materials were introduced into the root canal space,and the change of pH was recorded in the medium surrounding the root.

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Their findings indicated that bleaching materials, unlike calcium hydroxide, can diffuse readily through dentin. Older teeth and ones with smaller dentin tubule diameter are less permeable than young teeth and those with larger tubular diameter.16 The presence of cementum covering the radicular dentin decreases fluid flow across the dentin tubules.16 An increase of the dentinal surface area exposed and the reduction of dentin thickness increases its permeability.i7 The cervical region of the root was found to be more permeable than the middle, whereas the apical area was reported to be highly impermeable.18-20 Pretreatment of dentin or cementum with different agents may alter their permeability capacity.14, 19-21 Acid etching and materials such as 5% chloramine, ammoniacal silver nitrate, ethylenediaminetetraacetic acid (EDTA), 3% Hz02 and 5.25% sodium hypochlorite 19,2o were found to increase dentin permeability. 14$21 Smear layer was found to account for up to 85% resistance to fluid movement across human dentin.22 The increase of temperature increases dentin permeability.17l2’ Pashley et a1.21 found that an increase of 40” C almost doubled the fluid llow across untreated dentin. They concluded that this increment is due to thermal expansion, inducing increases in tubular diameter. To achieve the best esthetic results from bleaching, the clinician often performs several procedures that simultaneously facilitate penetration of the oxidizing agents to the tissues surrounding the root. These procedures usually include freshening of the dentin tubules, removal of the smear layer, the use of acid etchants, and the application of heat. In this study defects were simulated along the circumferential line of the CEJ. It is generally believed that in 10% of the cases the cementum and enamel do not meet at the CEJ, thus leaving exposed dentin tubules.23 This concept was based on old studies done almost a century ago, often subjected to errors in ground-section microscopy. Recent studies have indicated that cementum-enamel relationship at the CEJ is dynamic and may vary among different groups of teeth in the same person and even in the same tooth 24*25 Schroeder and Scherle25 found that the four aspects of the same tooth had different CEJ characteristics. The dentin could be exposed on one side of the tooth whereas on the other sides it could be fully covered with cementum. Dentin exposure occurred more on the buccal and the distal sides. Dentin exposure in premolars is very rare, ranging up to 4%, whereas in anterior teeth it may occur in 25% of the cases.24,25 In this study model, premolar teeth were used for three principal reasons: (1) their availability and the

Penetrance of hydrogen peroxide during bleaching


possibility to obtain a large sample of intact teeth, (2) the small incidence of preexisting dentin exposures at the CEJ, and (3) the young age of the patients, because most reports of bleaching-related root resorption have been associated with young adults. Under the conditions of this study it was found that human tooth permeability to 30% Hz02 during bleaching may reach up to 82% of the total volume applied. The variations in tooth permeability among the teeth tested could be due to morphologic and chemical variations and to the difference in the remaining dentin thickness. In this study bleaching was performed with the minimum quantity of Hz02 usually used for thermocatalytic bleaching. This resulted in more than 83,000 nmol of Hz02 penetration. The ability of Hz02 and other oxygen radicals to cause cellular and tissue distruction is a known phenomenon. 26-29 Simon et a1.27 found that Hz02 caused irreversible damage to human fibroblasts. They speculated that because Hz02 is a small uncharged molecule, it could cross cell membranes easily. There could be two general mechanisms by which Hz02 might kill cells27: (I) by penetrating the cell and damaging a vital internal component after exhausting the protective mechanisms or (2) by causing a fatal lesion to the external cell membrane, which is relatively unprotected. Fligiel et a1.28found that amounts of 50 to 5000 nmol Hz02 increased the susceptibility of hemoglobin to proteolysis by a number of enzymes. Ramp et a1.29 found that small amounts of Hz02 inhibited glucose metabolism and collagen synthesis and decrease bone weight and alkaline phosphatase activity. SUMMARY


Twenty-two extracted human teeth were tested for Hz02 penetration during internal thermocatalytic bleaching. The quantification of Hz02 penetration was performed directly with a specific assay. All teeth showed a certain permeability to 30% HzOz, which could reach up to 82% of the total volume applied. The study model suggested here may serve for examining various factors affecting Hz02 penetration through dentin and cementum during bleaching. I extend my gratitude to Drs. Uzi Rudolfson and Lucille Rotstein for their help in obtaining the teeth for this study. REFERENCES

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WWY, Cohen S, Borer RF. External cervical root resorption following bleaching. J Endod 1986;12:414-8. 7. Friedman S, Rotstein I, Libfeld H, Stabholz A, Heling I. Incidence of external root resorption and esthetic results in 58 bleached oulnless teeth. Endod Dent Traumatol 1988:4:23-6. 8. Friedman* S.‘Surgical-restorative treatment of bleaching related external root resorption. Endod Dent Traumatol 1989;5: 63-7. 9. Madison

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ORAL PATHOL November 1991

EC, Hauptfuehrer

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Hampson EL, Atkinson AM. The relation between drugs used in root canal therapy and the permeability of the dentin. Br Dent J 1964;116:546-50. Marshall FJ, Massler M, Dute HL. Effects of endodontic treatments on permeability of root dentin. ORAL SURG ORAL MED ORAL PATHOL 1960;13:208-23. Pashley DH, Thompson SM, Stewart FP. Dentin permeability: effects of temperature on hydraulic conductance. J Dent Res 1983;62:956-9. Pashley DH, Livingston MJ, Greenhill JD. Regional resistance to fluid flow in human dentin, in vitro. Arch Oral Biol 1978;23:807-10. Bhaskar SN. Orban’s oral histology and embryology. 8th ed. St Louis: CV Mosby, 1976:195. Muller CJF, Van Wyk CW. The amelo-cemental junction. J Dent Assoc S Afr 1984;39:799-803. Schroeder HE, Scherle WF. Cemento-enamel junctionrevisited. J Periodont Res 1988;23:53-9. Halliwell B, Gutteridge JMC. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;2 19: I- 14. Simon RH, Scoggin CH, Patterson D. Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals. J Biol Chem 1981;256:718 l-6. Fligiel SEG, Lee EC, McCoy JP, Johnson KJ, Varani J. Protein degradation following treatment with hydrogen peroxide. Am J Path01 1984;115:418-25. Ramp WK, Arnold RR, Russell JE, Yancey JM. Hydrogen peroxide inhibits glucose metabolism and collagen synthesis in bone. J Periodontol 1987;58:340-4.

Reprint requests: I. Rotstein, CD Department of Endodontics Hadassah Faculty of Dental P.O. Box 1172 Jerusalem 91010, Israel


In vitro determination and quantification of 30% hydrogen peroxide penetration through dentin and cementum during bleaching.

Bleaching pulpless teeth with 30% hydrogen peroxide was reported to cause external cervical root resorption. It was hypothesized that H2O2 penetrated ...
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