- Email: [email protected]
Effect of reduced air pressure on dye penetration in standardized voids
Effect of reduced air pressure on dye penetration in standardized voids Reza B. Kazemi, DMD, a and Larz S.W. SpSngberg, DDS, PhD, b Farmington, Conn. DEPARTMENT OF RESTORATIVEDENTISTRYAND ENDODONTOLOGY,UNIVERSITYOF CONNECTICUTHEALTHCENTER, SCHOOLOF DENTALMEDICINE
Objective. This
study determined the correlation between the degree of dye penetration in standardized voids to various reduced pressures. Study design. Standardized voids of 0.50 and 0.22 mm in diameter were created in 60 human roots; specimens were divided into five groups. Four groups were subjected to a 2% filtered aqueous methylene blue dye solution under different conditions of atmospheric pressure for 7 days and reduced pressures of a 10 torr, a 260 torr, and a 510 torr for 30 minutes. The conditions for the fifth group were similar to the last group except that ethyl alcohol was substituted for water in the dye. Results. All positive control specimens showed complete dye penetration. The least linear dye penetration was observed in passively immersed specimens. The results were significantly different from those when reduced pressure had been used (p < 0.001). There were no significant differences among the groups with reduced pressure. No significant difference was found at any pressure level between the means of dye diffusion in the 0.50 and the 0.22 mm voids. The smallest standard deviations were observed for the most reduced air pressure of 10 torr, followed by the group of the least reduced pressure of 510 torr, with the use of the tincture dye solution. Conclusions. A lO-torr reduced pressure and an application of the tincture dye solution would provide an improved method for microleakage studies. (ORAL SURG ORAL MED ORAL PAIHOL ORAL RADIOL ENDOD 1995;80:720-5)
Recently, Wu and Wesselink 1 noted that more than 20% of scientific articles presented in the leading endodontic journals were leakage studies. They observed that uncertainty exists with respect to methods. They further suggested that it would be more valuable to focus on methods for leakage studies than on outcomes of studies of leakage with still unrefined techniques. It is still unclear to what extent leakage of a tracer such as dye observed in experiments in vitro is clinically relevant. This may be because the results obtained from such experiments are incorrectly interpreted or misapplied. It is clearly not appropriate to attempt to correlate tracer leakage with radiographically detectable imperfections of a root canal filling; the radiographic technique p e r s e has insufficient resolution.2, 3 Because of the complex interrelationship between multiple factors that affect the outcome of endodontic treatment, it is virtually impossible to study the correlation between tracer leakage and Presented at the Second Annual Connecticut Symposiumon Endodontic Biology. aAssistant Professor. bprofessor and Head. Received for publication Apr. 4, 1995; returned for revision May 5, 1995 and June 19, 1995; accepted for publication July 12, 1995. Copyright 9 1995 by Mosby-Year Book, Inc. 1079-2104/95/$5.00 + 0 7/15/68044
720
treatment results. Such experiments would require an unreasonable large sample size. Success of root canal treatment is related to the reduction or elimination of bacteria cells or microbial byproducts. 4-7 The quality of the obturation is one of many steps in this process of reducing microbial irritation. In the process of developing new obturation materials and improved techniques, leakage studies with tracers such as dye are therefore useful in evaluating adhesive properties. Consequently, it is essential that optimal standardized methods to study adhesive properties of obturatRm materials are developed. Such methods are clinically relevant and very useful for material evaluation if they have a high degree of reliability with fewer chances for experimental errors. No such generally accepted highly reliable method to study the quality of adhesion of endodontic filling material is yet available. Sp~ngberg et al. 8 made the observation that the literature on dye leakage contains conflicting results often with very high standard deviations. Voids associated with defective root canal obturation are generally shaped like a capillary and are long and narrow. Dye penetration into small voids would therefore be by capillary forces 1' 9-10 created by the collapse pressure at the liquid-air interface. In 1982, Spradling and Senia 11 observed that dye solution did not penetrate all the way through known voids in roots and
ORAL SURGERY ORAL MEDICINEORAL PATHOLOGY Volume 80, Number 6 suggested that entrapped air may have hindered the dye from penetrating. Spgngberg et al., 8 Goldman et al.,12 and Wu et al. 13 in different experiments demonstrated that the hypothesis of Spradling and Senia 11 was correct. Unless air is evacuated from a capillary void, complete dye penetration by capillary force will not occur until a balance has been established between the collapse pressure and the surface tension of the fluid. In a follow-up study in 1991, Oliver and Abbott 14 concluded that dye studies without attempts to evacuate entrapped air are "unreliable and very variable." Although more than 10 years have passed since Spradling and Senia 11 made their observation and their theory repeatedly has proven to be right, a' 12-14 passive dye penetration studies are published in an unending stream. There are some publications with different views on the need to apply reduced pressure during dye leakage studies. 15-16 These studies, however, fail to establish a protocol with known experimental conditions. Nevertheless, the interesting conclusion of one showed that there were significant differences between the results of dye penetration for positive control groups as well as for a tested material (IRM) when reduced pressure had been applied. 15 It is essential to deliberately incorporate some degree of well-defined leakage in the specimens when comparing different leakage models.13 Furthermore, different methods of dye penetration in small voids with unknown size and form and with few specimens will not provide comparable reliable results; the standard deviation consistently has proven to be large in these types of experiments. Most studies applying reduced pressure have used an absolute pressure of approximately 25 torr. Empirically this level of evacuation appears sufficient for practical experimentation. One study used an absolute pressure as low as 100 mtorr.8 Although the relevance of dye penetration studies may be questionable, they serve specific purposes provided they are performed with optimal techniques. However, there are no correlations shown between the high degree of dye leakage and endodontic failures, and no correlation has been established between clinically and radiographically visible voids and high level of dye leakage. For the selected situations when dye penetration examinations are useful it is important that more basic information about this technique is obtained. A diverse range of dye techniques to improve the assessment of microleakage have been introduced.i, 8, 13-21 Among them, the methylene blue dye penetration technique is the most popular method particularly in endodontic studies. J, 8.18-19, 21 Methylene blue dye (C~6HlsCIN3S) is very soluble in water
Kazemi and Spdngberg
721
and easily penetrates the water compartment of the dental structure; however, it does not absorb to the inorganic dental matrix or apatite crystals and its intensity appears to be relatively uniform throughout the canals. 18 Water is the common solvent for methylene blue when used for leakage studies. Our observation in several experiments has been that the water solution easily drains from filled voids during the processing of the teeth to visualize the depth of dye penetration. Therefore little dye remains in some cases to visually track the original penetration. In pilot experiments methylene blue in alcohol has proven to stain the voids better than aqueous solutions even when much of the fluid dye had drained. The purpose of this study was to evaluate the degree of dye penetration in known voids at various reduced pressures with the use of water or alcohol as dye solvents. The purpose was not to evaluate the relevance of dye penetration studies. MATERIAL AND METHODS Circular roots of single- and multi-rooted fresh human teeth were collected. They were examined under a surgical telescope at x2.3 found to be free of any visible cracks, resorptions, and decalcifications and subsequently stored in 1% sodium hypochlorite (pH = 11.63) for 72 hours. The teeth were then transferred and stored in a 0.02% sodium azide solution (pH--7.15) until the experiments were begun. The coronal sections of the teeth were cut 2 mm below the cementoenamel junction. The apical 2 mm of all roots were also resected to decrease possible effects of apical branching of the root canals. The roots in the various groups did not differ significantly in length (mean range, 9.98 -+ 0.72 mm to 10.48 -+ 0.46 mm). Canal preparation was performed with Gates-Glidden burs nos. 3 through 6. Roots with canals larger than no. 6 were discarded. The canals were irrigated with 5.25% sodium hypochlorite (pH = 7.95) during preparation to remove debris. When the preparation was finished, the canals were rinsed thoroughly with distilled water. The prepared root canals were dried with ethyl alcohol (97%) followed by pressured air for 30 seconds. An identification dent was notched with a diamond disk on one side of the root. Two orthodontic stainless steel ligature wires with different diameters (0.50 mm and 0.22 mm) were inserted into each canal with the larger wire placed on the notched side of the root. The wires had a minimum of 5 mm protruding from either end of the root sample. The wires were carefully stabilized with sticky wax against the root canal walls at each side of the canals to ensure that there were no connections between the two wires. The
722
A
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 1995
Kazemi and Spdmgberg
AH26 Notch Small Void
Large Void
Dentin Nail Polish
Sticky Wax
Orthodontic Wires
Group 1 mB ,==
WQ
tt
B
each group had two extra specimens as positive controis. Therefore, in total, 60 root specimens were prepared for experimental and control roots. Without cutting off the protruding wires in the coronal end in the control specimens, the roots were painted with two layers of nail polish and covered with a thick layer of sticky wax. The wires were then pulled out of the canals, leaving continuous voids open in both root ends.
Coronal End
Apical End
Cylinder Fig. 1. Schematic review of experimental procedures. A, Preparation of specimen/voids. B, Serial dentin wafer (disk) cutting to assess dye penetration in voids. apical end of the canals were sealed with sticky wax with care to prevent any wax from entering the canals. The specimens were positioned apex down in utility wax. AH26 sealer (DeTrey, Zurich, Switzerland) was mixed according to the manufacturer's directions and carefully introduced into the root canal space with a no. 3 paste filler lentulo spiral (Union Broach, Emigsville, Pa.). The specimens were set at room temperature (72 + 1~ F) for 72 hours and then radiographed. Roots with any radiographic evidence of incomplete obturation were discarded. The wires in the coronal end of each canal were carefully cut off and smoothed with a polishing disk. The external surface of the roots were painted with two layers of nail polish, and when dried, they were covered with a thick layer of sticky wax. The wires were then withdrawn in apical direction from the specimens to create standardized voids closed in the coronal end (Fig. 1, A). Fifty specimens were then randomly divided into five experimental groups of 10 roots each. In addition,
Specimens were immersed for 7 days at atmospheric pressure (101.32 kPa/760 torr) in 100 ml of filtered (0.22 ~m) 2% aqueous methylene blue dye solution (pH 3.50) and incubated at 37 ~ C and 100% humidity. The specimens were recovered from the dye solution, the apical ends of the voids sealed with sticky wax, and then the specimens were rinsed under running water for 5 seconds. With the exception of the apical end, the sticky wax and nail polish were peeled off with a razor blade. The external surface of the roots and the coronal end of the voids were inspected in a stereomicroscope (x4 to x25.2 magnifications; Nikon SMZ-2T Japan) for any sign of dye penetration. Specimens that showed no evidence Of dye penetration through the coronal end of the voids were mounted in a thin cylinder of transparent coldcuring resin (Leco Corporation, St. Joseph, Mich.) leaving the apical and coronal ends of the specimens free (Fig. 1, B). Sectioning of the specimens was begun from the coronal ends using an Isomet saw (Buehler, Lake Bluff, Ill.). After the initial observation of the uncut coronal root surface, the diamond wafering blade (Buehler) was set to prepare the dentin disks with interval sections of 0.4 mm. As soon as any sign of dye penetration through either void was observed, the remaining length of the root was measured with a digitized micrometer with accuracy of 0.001 mm (Mitutoyo, Japan). The measurements were repeated twice and the average was recorded. This process was continued until a sign of dye penetration in the second void was observed. The collected data were converted to percentage to allow comparisons. An analysis of variance (one-way A N O V A ) and a t test were also performed to analyze the data.
Group 2 The specimens were placed in a vacuum flask, immersed in 100 ml of filtered (0.22 pm) 2% aqueous methylene blue dye solution (pH 3.50). Air was evacuated to a reduced pressure of 1.33 kPa (10 torr) and maintained for 30 minutes. The remaining parts of the experiment were carried out similar to group 1.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Kazemi and Sp~ngberg
723
Volume 80, Number 6
o~ C
tO0
0
~
8o
~
60
@l ,~
40
e.J
o ffl i-
2o
~
o
760 Torr 10 Torr 260 Torr 510 Torr 510 Tort a.d. a.d. a.d. a.d. t.d.
9 LargeVoid (0.50 mm) []
Applied Pressures
Small Void (0.22 mm )
a.d.: Aqueous Dye Solution i.d.: Tincture Dye Solution
Fig. 2. Dye penetration in voids under various experimental conditions.
Table I. Dye penetration in percentages of total length of voids Group Large voids (0.50 ram) 2 5 3 4 1 Small voids (0.22 ram) 2 5 3 4 1
Mean
SD
10 t o r t - a q u e o u s solution 510 t o r r - t i n c t u r e solution 260 t o r r - a q u e o u s solution 510 t o r r - a q u e o u s solution 760 torr-aqueous solution
99.81 99.52 97.06 93.39 19.47
0.39 1.53 8.32 12.50 12.11
NS NS NS NS *
10 t o r r - a q u e o u s solution 510 t o r r - t i n c t u r e solution 260 t o r r - a q u e o u s solution 510 t o r r - a q u e o u s solution 760 ton--aqueous solution
98.86 96.81 97.84 86.50 13.90
2.78 3.96 5.58 18.91 12.04
NS NS NS NS *
Group 1: Atmospheric/passive air pressure. Groups 2, 3, 4, 5: Reduced air pressure. SD, Standard deviation. NS, No significant differences among groups of reduced air pressure. *Significant differences, between group 1 and all groups of reduced air pressure (p < 0.001).
Group 3 The experiment was similar to group 2 except that pressure was reduced to 34.66 kPa (260 torr).
Group 4 The experiment was similar to group 2 except that the pressure was reduced to 67.99 kPa (510 ton'). Group 5 The experiment was similar to group 4. Ethyl alcohol (97%) was substituted for water in the methylene blue dye solution.
RESULTS All positive control specimens demonstrated dye penetration through the entire voids. The means and standard deviations of the results of dye penetration under the five different laboratory conditions are summarized in Table I and Fig. 2. There was not a statistically significant difference in the overall length of the root specimens (p = 0.22). The least dye penetration was observed in passively immersed (no vacuum) specimens with no case of complete penetration. Pressure levels did not show a significant difference between the means of dye dif-
724
Kazemi and Spdmgberg
fusion in the 0.22- and the 0.50-mm voids. There were significant differences (p < 0.001) (Table I) between passive/atmospheric pressure dye penetration at either void size and dye diffusion at corresponding void size at any reduced air pressure. The smallest standard deviations were observed for the most reduced air pressure of 10 torr, followed by the group of reduced pressure of 510 torr with the tincture dye solution (Table I and Fig. 2). The largest standard deviations were found for the reduced pressure of 510 torr group in the presence of aqueous dye solution followed by atmospheric pressure experiment. DISCUSSION It has been shown that the passive immersing technique is not a reliable method to assess dye penetration in voids interfacing dental materials and dentin.8, 13, 14 Entrapped air should be removed to allow the dye to totally penetrate in the space between the dentin wall and the dental material. The aim o f this study was to gather more relevant information to determine whether the range of reduced air pressure would have an impact on the degree of entrapped air removal and consequently on the completion of dye penetration through the standardized/known voids. To reduce the discrepancies among the root specimens, attention was paid to select roots of similar overall length (p = 0.22). Furthermore, as suggested by Wn and Wesselink, 1 a pilot study was conducted to determine whether the methylene blue dye solution at a low pH was able to damage the tooth. Results indicated that the methylene blue dye solution used in this study did not have a significant effect on the integrity of the specimens. Methylene blue is a very weak acid and was easily neutralized during these experimental conditions. The application of atmospheric/passive pressure for 7 days showed the least dye penetration in both sizes of premeasured voids (group 1 in Table I and Fig. 2). On the other hand, all other groups regardless of the degree of reduced pressure exhibited complete or near complete dye penetration when air was evacuated (p < 0.001), (Table I). The findings support the results of previous studies by Spgmgberg et al., 8 Goldman et al., 12 Wu et al., 13 and Oliver and AbbottJ 4 In earlier pilot experiments methylene blue dye in alcohol had proven to stain the walls of the voids better than aqueous solution. It is our experience that the stain remains more permanent and is difficult to wash out even when much of the fluid dye has drained. On the other hand, if aqueous dye solution remains in the void, a more careful technique will be essential for sectioning of the specimens to avoid spreading the tracer to areas where dye had not penetrated. 18 The
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 1995
tincture dye solution did demonstrate a better penetration in both size voids. This is reflected in significantly smaller standard deviations (Table I) for the dye tincture results compared with the results from a similar experiment with aqueous solutions. This suggests that the use of dye tincture may provide more accurate results than aqueous dye solution at moderately reduced pressure. In conclusion, the statistical analysis of data in the present study revealed that: 1. Entrapped air did have a significant impact on the dye penetration in the voids. 2. Even minimum air pressure reduction allowed the dye to extensively penetrate the voids. 3. Standard deviation of the results is directly related to the pressure reduction used. 4. It has been suggested that the volume of the void has an important impact on the dye diffusion. However, the results of the present study showed that there were no significant differences in linear dye penetration between the two different sizes of voids used in the experiments. These findings are in support of previous findings 8 that the volume of void does not have a significant effect on dye penetration when the diameter of void is 0.5 m m or smaller. Greater dye penetration does not indicate a wider void. It is greatly dependent on the presence or absence of entrapped air or liquid in the void. 1 5. Alcohol-based dye solution demonstrated a better and m o r e reliable staining of the voids than the aqueous dye solution. We suggest that a 10 torr reduced pressure and an application of the tincture dye solution would provide the most improved method for microleakage studies. REFERENCES
1. Wu M-K, Wesselink PR. Endodontic leakage studies reconsidered: Part I. methodology,application, and relevance. Int Endodon J 1993;26:37-43. 2. Kersten HW, Wesselink PR, ThodenVan Velzen SK. The diagnostic reliability of the buccal radiograph after root canal filling. Int Endodon J 1987;20:20-4. 3. Friedman S, Rotstein I, Koren L, Trope M. Dye leakage in retrofilled dog teeth and its correlation with radiographic healing. J Endodon 1991;17:392-5. 4. Nair PNR, Sj6gren U, Krey G, Kahnberg K-E, Sundqvist G. Intraradicular bacteria and fungi in root-filled asymptomatic human teeth with therapy-resistant periapical lesions: a longterm light and electron microscopefollow-up study. J Endodon 1990;16:580-8. 5. BystrOmA, Happonen R-P, Sj6grenU, SundqvistG. Healing of periapical lesions of pulpless teeth after endodontic treatment with controlledasepsis. EndodonDent Traumatol 1987; 3:58-63. 6. Engstr6m B, Lundberg M. The correlation between positive culture and the prognosis of root canal therapy after pulpectomy. Odont Rev 1965;16:194-203.
Kazemi and Spdngberg
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
725
Volume 80, Number 6 7. EngstrOm B, Hhrd AF, Segerstad L, Ramstr6m G, Frostell G. Correlation of positive cultures with the prognosis for root canal treatment. Odont Rev 1964;15:257-70. 8. Sp~ngberg LSW, Acierno TG, Cha BY. Influence of entrapped air on the accuracy of leakage studies using dye penetration methods. J Endodon 1989;15:548-51. 9. Schnell F. Effect of immediate dowel space preparation on the apical seat of endodontically filled teeth. ORAL SURG ORAL MED ORAL PATHOL 1978;45:470-4. 10. Pitt Ford TR. The leakage of root fillings using glass ionomer cement and other materials. Br Dent J 1979;146:273-8. 11. Spradling PM, Senia S. The relative sealing ability of pastetype filling materials. J Endodon 1982;8:543-9. 12. Goldman M, Simmonds S, Rush R. The usefulness of dye penetration studies reexamined. ORALSURGORALMED ORAL PATHOL 1989;67:327-32. 13. Wu M-K, De Gee A J, Wesselink PR. Fluid transport and dye penetration along root canal fillings. Int Endodon J 1994; 27:233-8. 14. Oliver CM, Abbott PV. Entrapped air and its effects on dye penetration of voids. Endodon Dent Traumatol 1991 ;7:135-8. 15. Peters LB, Harrison JW. A comparison of leakage of filling materials in demineralized and non-demineralized resected root ends under vacuum and nonvacuum conditions. Int Endodon J 1992;25:273-8.
16. Dickson SS, Peters DD. Leakage evaluation with and without vacuum of two gutta-percha fill techniques. J Endodon 1993;19:398-403. 17. Taylor MJ, Lynch E. Microleakage. J Dent 1992;20:3-10. 18. Matloff IR, Jensen JR, Singer L, Tabibi A. A comparison of methods used in root canal sealability studies. ORAL SURG ORAL MED ORAL PATHOL 1982;53:203-8. 19. Starkey DL, Anderson RW, Pashley DH. An evaluation of the effect of methylene blue dye pH on apical leakage [Abstract #28]. J Endodon 1993;19:192. 20. Dalat DM, Sp~kngberg LSW. Comparison of apical leakage in root canals obtul-ated with various gutta-percha techniques using a dye vacuum tracing method. J Endodon 1994; 20:315 9. 21. A1-Ghamdi A, Wennberg A. Testing of sealing ability of endodontic filling materials. Endod Dent Traumatol t994;10: 249-55.
Reprint requests: Reza Kazemi, DMD Department of Restorative Dentistry and Endodontology University of Connecticut Health Center School of Dental Medicine 263 Farmington Avenue Farmington, CT 06030-1715
Objective. This
study determined the correlation between the degree of dye penetration in standardized voids to various reduced pressures. Study design. Standardized voids of 0.50 and 0.22 mm in diameter were created in 60 human roots; specimens were divided into five groups. Four groups were subjected to a 2% filtered aqueous methylene blue dye solution under different conditions of atmospheric pressure for 7 days and reduced pressures of a 10 torr, a 260 torr, and a 510 torr for 30 minutes. The conditions for the fifth group were similar to the last group except that ethyl alcohol was substituted for water in the dye. Results. All positive control specimens showed complete dye penetration. The least linear dye penetration was observed in passively immersed specimens. The results were significantly different from those when reduced pressure had been used (p < 0.001). There were no significant differences among the groups with reduced pressure. No significant difference was found at any pressure level between the means of dye diffusion in the 0.50 and the 0.22 mm voids. The smallest standard deviations were observed for the most reduced air pressure of 10 torr, followed by the group of the least reduced pressure of 510 torr, with the use of the tincture dye solution. Conclusions. A lO-torr reduced pressure and an application of the tincture dye solution would provide an improved method for microleakage studies. (ORAL SURG ORAL MED ORAL PAIHOL ORAL RADIOL ENDOD 1995;80:720-5)
Recently, Wu and Wesselink 1 noted that more than 20% of scientific articles presented in the leading endodontic journals were leakage studies. They observed that uncertainty exists with respect to methods. They further suggested that it would be more valuable to focus on methods for leakage studies than on outcomes of studies of leakage with still unrefined techniques. It is still unclear to what extent leakage of a tracer such as dye observed in experiments in vitro is clinically relevant. This may be because the results obtained from such experiments are incorrectly interpreted or misapplied. It is clearly not appropriate to attempt to correlate tracer leakage with radiographically detectable imperfections of a root canal filling; the radiographic technique p e r s e has insufficient resolution.2, 3 Because of the complex interrelationship between multiple factors that affect the outcome of endodontic treatment, it is virtually impossible to study the correlation between tracer leakage and Presented at the Second Annual Connecticut Symposiumon Endodontic Biology. aAssistant Professor. bprofessor and Head. Received for publication Apr. 4, 1995; returned for revision May 5, 1995 and June 19, 1995; accepted for publication July 12, 1995. Copyright 9 1995 by Mosby-Year Book, Inc. 1079-2104/95/$5.00 + 0 7/15/68044
720
treatment results. Such experiments would require an unreasonable large sample size. Success of root canal treatment is related to the reduction or elimination of bacteria cells or microbial byproducts. 4-7 The quality of the obturation is one of many steps in this process of reducing microbial irritation. In the process of developing new obturation materials and improved techniques, leakage studies with tracers such as dye are therefore useful in evaluating adhesive properties. Consequently, it is essential that optimal standardized methods to study adhesive properties of obturatRm materials are developed. Such methods are clinically relevant and very useful for material evaluation if they have a high degree of reliability with fewer chances for experimental errors. No such generally accepted highly reliable method to study the quality of adhesion of endodontic filling material is yet available. Sp~ngberg et al. 8 made the observation that the literature on dye leakage contains conflicting results often with very high standard deviations. Voids associated with defective root canal obturation are generally shaped like a capillary and are long and narrow. Dye penetration into small voids would therefore be by capillary forces 1' 9-10 created by the collapse pressure at the liquid-air interface. In 1982, Spradling and Senia 11 observed that dye solution did not penetrate all the way through known voids in roots and
ORAL SURGERY ORAL MEDICINEORAL PATHOLOGY Volume 80, Number 6 suggested that entrapped air may have hindered the dye from penetrating. Spgngberg et al., 8 Goldman et al.,12 and Wu et al. 13 in different experiments demonstrated that the hypothesis of Spradling and Senia 11 was correct. Unless air is evacuated from a capillary void, complete dye penetration by capillary force will not occur until a balance has been established between the collapse pressure and the surface tension of the fluid. In a follow-up study in 1991, Oliver and Abbott 14 concluded that dye studies without attempts to evacuate entrapped air are "unreliable and very variable." Although more than 10 years have passed since Spradling and Senia 11 made their observation and their theory repeatedly has proven to be right, a' 12-14 passive dye penetration studies are published in an unending stream. There are some publications with different views on the need to apply reduced pressure during dye leakage studies. 15-16 These studies, however, fail to establish a protocol with known experimental conditions. Nevertheless, the interesting conclusion of one showed that there were significant differences between the results of dye penetration for positive control groups as well as for a tested material (IRM) when reduced pressure had been applied. 15 It is essential to deliberately incorporate some degree of well-defined leakage in the specimens when comparing different leakage models.13 Furthermore, different methods of dye penetration in small voids with unknown size and form and with few specimens will not provide comparable reliable results; the standard deviation consistently has proven to be large in these types of experiments. Most studies applying reduced pressure have used an absolute pressure of approximately 25 torr. Empirically this level of evacuation appears sufficient for practical experimentation. One study used an absolute pressure as low as 100 mtorr.8 Although the relevance of dye penetration studies may be questionable, they serve specific purposes provided they are performed with optimal techniques. However, there are no correlations shown between the high degree of dye leakage and endodontic failures, and no correlation has been established between clinically and radiographically visible voids and high level of dye leakage. For the selected situations when dye penetration examinations are useful it is important that more basic information about this technique is obtained. A diverse range of dye techniques to improve the assessment of microleakage have been introduced.i, 8, 13-21 Among them, the methylene blue dye penetration technique is the most popular method particularly in endodontic studies. J, 8.18-19, 21 Methylene blue dye (C~6HlsCIN3S) is very soluble in water
Kazemi and Spdngberg
721
and easily penetrates the water compartment of the dental structure; however, it does not absorb to the inorganic dental matrix or apatite crystals and its intensity appears to be relatively uniform throughout the canals. 18 Water is the common solvent for methylene blue when used for leakage studies. Our observation in several experiments has been that the water solution easily drains from filled voids during the processing of the teeth to visualize the depth of dye penetration. Therefore little dye remains in some cases to visually track the original penetration. In pilot experiments methylene blue in alcohol has proven to stain the voids better than aqueous solutions even when much of the fluid dye had drained. The purpose of this study was to evaluate the degree of dye penetration in known voids at various reduced pressures with the use of water or alcohol as dye solvents. The purpose was not to evaluate the relevance of dye penetration studies. MATERIAL AND METHODS Circular roots of single- and multi-rooted fresh human teeth were collected. They were examined under a surgical telescope at x2.3 found to be free of any visible cracks, resorptions, and decalcifications and subsequently stored in 1% sodium hypochlorite (pH = 11.63) for 72 hours. The teeth were then transferred and stored in a 0.02% sodium azide solution (pH--7.15) until the experiments were begun. The coronal sections of the teeth were cut 2 mm below the cementoenamel junction. The apical 2 mm of all roots were also resected to decrease possible effects of apical branching of the root canals. The roots in the various groups did not differ significantly in length (mean range, 9.98 -+ 0.72 mm to 10.48 -+ 0.46 mm). Canal preparation was performed with Gates-Glidden burs nos. 3 through 6. Roots with canals larger than no. 6 were discarded. The canals were irrigated with 5.25% sodium hypochlorite (pH = 7.95) during preparation to remove debris. When the preparation was finished, the canals were rinsed thoroughly with distilled water. The prepared root canals were dried with ethyl alcohol (97%) followed by pressured air for 30 seconds. An identification dent was notched with a diamond disk on one side of the root. Two orthodontic stainless steel ligature wires with different diameters (0.50 mm and 0.22 mm) were inserted into each canal with the larger wire placed on the notched side of the root. The wires had a minimum of 5 mm protruding from either end of the root sample. The wires were carefully stabilized with sticky wax against the root canal walls at each side of the canals to ensure that there were no connections between the two wires. The
722
A
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 1995
Kazemi and Spdmgberg
AH26 Notch Small Void
Large Void
Dentin Nail Polish
Sticky Wax
Orthodontic Wires
Group 1 mB ,==
WQ
tt
B
each group had two extra specimens as positive controis. Therefore, in total, 60 root specimens were prepared for experimental and control roots. Without cutting off the protruding wires in the coronal end in the control specimens, the roots were painted with two layers of nail polish and covered with a thick layer of sticky wax. The wires were then pulled out of the canals, leaving continuous voids open in both root ends.
Coronal End
Apical End
Cylinder Fig. 1. Schematic review of experimental procedures. A, Preparation of specimen/voids. B, Serial dentin wafer (disk) cutting to assess dye penetration in voids. apical end of the canals were sealed with sticky wax with care to prevent any wax from entering the canals. The specimens were positioned apex down in utility wax. AH26 sealer (DeTrey, Zurich, Switzerland) was mixed according to the manufacturer's directions and carefully introduced into the root canal space with a no. 3 paste filler lentulo spiral (Union Broach, Emigsville, Pa.). The specimens were set at room temperature (72 + 1~ F) for 72 hours and then radiographed. Roots with any radiographic evidence of incomplete obturation were discarded. The wires in the coronal end of each canal were carefully cut off and smoothed with a polishing disk. The external surface of the roots were painted with two layers of nail polish, and when dried, they were covered with a thick layer of sticky wax. The wires were then withdrawn in apical direction from the specimens to create standardized voids closed in the coronal end (Fig. 1, A). Fifty specimens were then randomly divided into five experimental groups of 10 roots each. In addition,
Specimens were immersed for 7 days at atmospheric pressure (101.32 kPa/760 torr) in 100 ml of filtered (0.22 ~m) 2% aqueous methylene blue dye solution (pH 3.50) and incubated at 37 ~ C and 100% humidity. The specimens were recovered from the dye solution, the apical ends of the voids sealed with sticky wax, and then the specimens were rinsed under running water for 5 seconds. With the exception of the apical end, the sticky wax and nail polish were peeled off with a razor blade. The external surface of the roots and the coronal end of the voids were inspected in a stereomicroscope (x4 to x25.2 magnifications; Nikon SMZ-2T Japan) for any sign of dye penetration. Specimens that showed no evidence Of dye penetration through the coronal end of the voids were mounted in a thin cylinder of transparent coldcuring resin (Leco Corporation, St. Joseph, Mich.) leaving the apical and coronal ends of the specimens free (Fig. 1, B). Sectioning of the specimens was begun from the coronal ends using an Isomet saw (Buehler, Lake Bluff, Ill.). After the initial observation of the uncut coronal root surface, the diamond wafering blade (Buehler) was set to prepare the dentin disks with interval sections of 0.4 mm. As soon as any sign of dye penetration through either void was observed, the remaining length of the root was measured with a digitized micrometer with accuracy of 0.001 mm (Mitutoyo, Japan). The measurements were repeated twice and the average was recorded. This process was continued until a sign of dye penetration in the second void was observed. The collected data were converted to percentage to allow comparisons. An analysis of variance (one-way A N O V A ) and a t test were also performed to analyze the data.
Group 2 The specimens were placed in a vacuum flask, immersed in 100 ml of filtered (0.22 pm) 2% aqueous methylene blue dye solution (pH 3.50). Air was evacuated to a reduced pressure of 1.33 kPa (10 torr) and maintained for 30 minutes. The remaining parts of the experiment were carried out similar to group 1.
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY
Kazemi and Sp~ngberg
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Volume 80, Number 6
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tO0
0
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o ffl i-
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760 Torr 10 Torr 260 Torr 510 Torr 510 Tort a.d. a.d. a.d. a.d. t.d.
9 LargeVoid (0.50 mm) []
Applied Pressures
Small Void (0.22 mm )
a.d.: Aqueous Dye Solution i.d.: Tincture Dye Solution
Fig. 2. Dye penetration in voids under various experimental conditions.
Table I. Dye penetration in percentages of total length of voids Group Large voids (0.50 ram) 2 5 3 4 1 Small voids (0.22 ram) 2 5 3 4 1
Mean
SD
10 t o r t - a q u e o u s solution 510 t o r r - t i n c t u r e solution 260 t o r r - a q u e o u s solution 510 t o r r - a q u e o u s solution 760 torr-aqueous solution
99.81 99.52 97.06 93.39 19.47
0.39 1.53 8.32 12.50 12.11
NS NS NS NS *
10 t o r r - a q u e o u s solution 510 t o r r - t i n c t u r e solution 260 t o r r - a q u e o u s solution 510 t o r r - a q u e o u s solution 760 ton--aqueous solution
98.86 96.81 97.84 86.50 13.90
2.78 3.96 5.58 18.91 12.04
NS NS NS NS *
Group 1: Atmospheric/passive air pressure. Groups 2, 3, 4, 5: Reduced air pressure. SD, Standard deviation. NS, No significant differences among groups of reduced air pressure. *Significant differences, between group 1 and all groups of reduced air pressure (p < 0.001).
Group 3 The experiment was similar to group 2 except that pressure was reduced to 34.66 kPa (260 torr).
Group 4 The experiment was similar to group 2 except that the pressure was reduced to 67.99 kPa (510 ton'). Group 5 The experiment was similar to group 4. Ethyl alcohol (97%) was substituted for water in the methylene blue dye solution.
RESULTS All positive control specimens demonstrated dye penetration through the entire voids. The means and standard deviations of the results of dye penetration under the five different laboratory conditions are summarized in Table I and Fig. 2. There was not a statistically significant difference in the overall length of the root specimens (p = 0.22). The least dye penetration was observed in passively immersed (no vacuum) specimens with no case of complete penetration. Pressure levels did not show a significant difference between the means of dye dif-
724
Kazemi and Spdmgberg
fusion in the 0.22- and the 0.50-mm voids. There were significant differences (p < 0.001) (Table I) between passive/atmospheric pressure dye penetration at either void size and dye diffusion at corresponding void size at any reduced air pressure. The smallest standard deviations were observed for the most reduced air pressure of 10 torr, followed by the group of reduced pressure of 510 torr with the tincture dye solution (Table I and Fig. 2). The largest standard deviations were found for the reduced pressure of 510 torr group in the presence of aqueous dye solution followed by atmospheric pressure experiment. DISCUSSION It has been shown that the passive immersing technique is not a reliable method to assess dye penetration in voids interfacing dental materials and dentin.8, 13, 14 Entrapped air should be removed to allow the dye to totally penetrate in the space between the dentin wall and the dental material. The aim o f this study was to gather more relevant information to determine whether the range of reduced air pressure would have an impact on the degree of entrapped air removal and consequently on the completion of dye penetration through the standardized/known voids. To reduce the discrepancies among the root specimens, attention was paid to select roots of similar overall length (p = 0.22). Furthermore, as suggested by Wn and Wesselink, 1 a pilot study was conducted to determine whether the methylene blue dye solution at a low pH was able to damage the tooth. Results indicated that the methylene blue dye solution used in this study did not have a significant effect on the integrity of the specimens. Methylene blue is a very weak acid and was easily neutralized during these experimental conditions. The application of atmospheric/passive pressure for 7 days showed the least dye penetration in both sizes of premeasured voids (group 1 in Table I and Fig. 2). On the other hand, all other groups regardless of the degree of reduced pressure exhibited complete or near complete dye penetration when air was evacuated (p < 0.001), (Table I). The findings support the results of previous studies by Spgmgberg et al., 8 Goldman et al., 12 Wu et al., 13 and Oliver and AbbottJ 4 In earlier pilot experiments methylene blue dye in alcohol had proven to stain the walls of the voids better than aqueous solution. It is our experience that the stain remains more permanent and is difficult to wash out even when much of the fluid dye has drained. On the other hand, if aqueous dye solution remains in the void, a more careful technique will be essential for sectioning of the specimens to avoid spreading the tracer to areas where dye had not penetrated. 18 The
ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY December 1995
tincture dye solution did demonstrate a better penetration in both size voids. This is reflected in significantly smaller standard deviations (Table I) for the dye tincture results compared with the results from a similar experiment with aqueous solutions. This suggests that the use of dye tincture may provide more accurate results than aqueous dye solution at moderately reduced pressure. In conclusion, the statistical analysis of data in the present study revealed that: 1. Entrapped air did have a significant impact on the dye penetration in the voids. 2. Even minimum air pressure reduction allowed the dye to extensively penetrate the voids. 3. Standard deviation of the results is directly related to the pressure reduction used. 4. It has been suggested that the volume of the void has an important impact on the dye diffusion. However, the results of the present study showed that there were no significant differences in linear dye penetration between the two different sizes of voids used in the experiments. These findings are in support of previous findings 8 that the volume of void does not have a significant effect on dye penetration when the diameter of void is 0.5 m m or smaller. Greater dye penetration does not indicate a wider void. It is greatly dependent on the presence or absence of entrapped air or liquid in the void. 1 5. Alcohol-based dye solution demonstrated a better and m o r e reliable staining of the voids than the aqueous dye solution. We suggest that a 10 torr reduced pressure and an application of the tincture dye solution would provide the most improved method for microleakage studies. REFERENCES
1. Wu M-K, Wesselink PR. Endodontic leakage studies reconsidered: Part I. methodology,application, and relevance. Int Endodon J 1993;26:37-43. 2. Kersten HW, Wesselink PR, ThodenVan Velzen SK. The diagnostic reliability of the buccal radiograph after root canal filling. Int Endodon J 1987;20:20-4. 3. Friedman S, Rotstein I, Koren L, Trope M. Dye leakage in retrofilled dog teeth and its correlation with radiographic healing. J Endodon 1991;17:392-5. 4. Nair PNR, Sj6gren U, Krey G, Kahnberg K-E, Sundqvist G. Intraradicular bacteria and fungi in root-filled asymptomatic human teeth with therapy-resistant periapical lesions: a longterm light and electron microscopefollow-up study. J Endodon 1990;16:580-8. 5. BystrOmA, Happonen R-P, Sj6grenU, SundqvistG. Healing of periapical lesions of pulpless teeth after endodontic treatment with controlledasepsis. EndodonDent Traumatol 1987; 3:58-63. 6. Engstr6m B, Lundberg M. The correlation between positive culture and the prognosis of root canal therapy after pulpectomy. Odont Rev 1965;16:194-203.
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Volume 80, Number 6 7. EngstrOm B, Hhrd AF, Segerstad L, Ramstr6m G, Frostell G. Correlation of positive cultures with the prognosis for root canal treatment. Odont Rev 1964;15:257-70. 8. Sp~ngberg LSW, Acierno TG, Cha BY. Influence of entrapped air on the accuracy of leakage studies using dye penetration methods. J Endodon 1989;15:548-51. 9. Schnell F. Effect of immediate dowel space preparation on the apical seat of endodontically filled teeth. ORAL SURG ORAL MED ORAL PATHOL 1978;45:470-4. 10. Pitt Ford TR. The leakage of root fillings using glass ionomer cement and other materials. Br Dent J 1979;146:273-8. 11. Spradling PM, Senia S. The relative sealing ability of pastetype filling materials. J Endodon 1982;8:543-9. 12. Goldman M, Simmonds S, Rush R. The usefulness of dye penetration studies reexamined. ORALSURGORALMED ORAL PATHOL 1989;67:327-32. 13. Wu M-K, De Gee A J, Wesselink PR. Fluid transport and dye penetration along root canal fillings. Int Endodon J 1994; 27:233-8. 14. Oliver CM, Abbott PV. Entrapped air and its effects on dye penetration of voids. Endodon Dent Traumatol 1991 ;7:135-8. 15. Peters LB, Harrison JW. A comparison of leakage of filling materials in demineralized and non-demineralized resected root ends under vacuum and nonvacuum conditions. Int Endodon J 1992;25:273-8.
16. Dickson SS, Peters DD. Leakage evaluation with and without vacuum of two gutta-percha fill techniques. J Endodon 1993;19:398-403. 17. Taylor MJ, Lynch E. Microleakage. J Dent 1992;20:3-10. 18. Matloff IR, Jensen JR, Singer L, Tabibi A. A comparison of methods used in root canal sealability studies. ORAL SURG ORAL MED ORAL PATHOL 1982;53:203-8. 19. Starkey DL, Anderson RW, Pashley DH. An evaluation of the effect of methylene blue dye pH on apical leakage [Abstract #28]. J Endodon 1993;19:192. 20. Dalat DM, Sp~kngberg LSW. Comparison of apical leakage in root canals obtul-ated with various gutta-percha techniques using a dye vacuum tracing method. J Endodon 1994; 20:315 9. 21. A1-Ghamdi A, Wennberg A. Testing of sealing ability of endodontic filling materials. Endod Dent Traumatol t994;10: 249-55.
Reprint requests: Reza Kazemi, DMD Department of Restorative Dentistry and Endodontology University of Connecticut Health Center School of Dental Medicine 263 Farmington Avenue Farmington, CT 06030-1715