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Solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue
JOURNAL OF ENDODONTICS I VOL 7, NO 10, OCTOBER 1981
Solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue Terence M. Gordon, DDS; Denise Damato, AS; and Paul Christner, PhD
Vital a n d necrotic b o v i n e t o o t h p u l p were e x p o s e d to 0, 1, 3, a n d 5% N a O C I for two to ten m i n u t e s . N a O C I (0%) h a d no dissolving p o w e r o n vital pulp, and small effect o n necrotic pulp. H o w e v e r , 3 a n d 5% N a O C 1 were e q u a l l y effective in dissolving a b o u t three f o u r t h s of the vital p u l p after t w o m i n u t e s ' exposure. N a O C 1 at 1, 3 a n d 5% were equally effective in dissolving 90% of the necrotic pulp after five m i n u t e s ' exposure.
Sodium hypochlorite has been used for many years as an adjunct to thorough mechanical instrumentation. A strongly alkaline chlorineliberating solution, its germicidal activity is related to the formation of hypochlorous acid on release of chlorine gas from solution.' Dakin-" reported the use of 0.5% of sodium hypochlorite solution to irrigate wounds during World War 1. Austin and Taylor 3 showed the solvent action of sodium hypochlorite (Dakin's solution) on nonvital tissue, noticing that the solution was only mildly inflammatory to normal tissue. Double strength chlorinated soda solution was first recommended empirically for use as a canal irrigant by Walker ~ in 1936. In an experimental study, Grossman and Meiman 5 in 1941 found double strength chlorinated soda solution (3% NaOCI) a most effective solvent, dissolving pulp tissue within 20 minutes to two hours. In the same year Grossman ~ recommended irrigation with hydrogen peroxide (3%) and sodium hypochlorite solution
466
(0.5%). The use of Clorox as a source of chlorinated soda or sodium hypochlorite was introduced by Lewis~ in 1954. In 1971, Senia and others s concluded that 5.25% sodium hypochlorite (Clorox) was not an effective solvent in canals with small diameters. The sodium hypochlorite solution was not used in conjunction with normal clinical procedures, but the solution was allowed to remain in the tooth for a specific time. In 1972, Lamers and others" showed that non-necrotic tissue fixed by paraformaldehyde dissolved more slowly in sodium hypochlorite solution. They also showed that 1% is less irritating and almost as effective as 5% sodium hypochlorite solution. Sp~mgberg and others'" in 1973 suggested the use of 0.5% sodium hypochlorite solution to minimize toxicity and maintain bacteriostatic effectiveness. However, at this dilution, the solvent effect was significantly diminished and was effective only on necrotic tissue. The cytotoxicity studies were based on results using Hela and I, cells. Trowbridge 1~
pointed out that care should be taken in interpreting these cytotoxic results. Baker and associates '~ evaluated various irrigants for their effectiveness in removing tooth canal debris. They found no difference between normal saline solution and 1% sodium hypochlorite solution. Citing the Sp~ngberg study, they recommended the use of saline solution as an irrigant because of its lack of toxicity. McComb and Smith 1'~used a scanning electron microscope and reported that virtually no debris was seen throughout the entire canal after 6% sodium hypochlorite solution was used with instrumentation. The 1% solution was not as effective. In pilot studies using 5% sodium hypochlorite solution, Rutberg and others" found extensive tissue damage, and the affected tissue was necrotised and dissolved. Svec and Harrison '~ showed that a combination of 5.25% sodium hypochlorite solution and 3% hydrogen peroxide was significantly more effective in cleaning the canal system at 1- and 3-mm apical levels. At the 5-mm level, normal saline solution was equally effective as an irrigant. Trepagnier and others TM reported a quantitative evaluation of different dilutions of sodium hypochlorite solution. A determination of the dissolution and removal of pulpal and dentinal debris from the canal was
JOURNAL OF ENDODONTICS I VOL 7, NO 10, OCTOBER 1981
measured by analyzing the hydroxyproline content of the solution that resulted after irrigation. Pulp tissue consists of 15% of collagen, containing approximately 13% hydroxyprolinc. The results indicated that sodium hypochlorite solution is a powerful solvent whose action starts immediately and continues for at least an hour. Dilution to 2.5% does not affect its solvent action appreciably, but a modified Dakin's solution has little solvent action. They, therefore, suggested the use of the diluted solution, that is, 2.5%. H a n d and others '7 evaluated the effect of diluting sodium hypochlorite solution on its solvent action. They exposed necrotic tissue to various concentrations of sodium hypochlorite solution, and the percentage of tissue weight changes was determined. Statistical analysis indicated that dilution of 5.25% sodium hypochlorite resulted in a significant decrease in its ability to dissolve necrotic tissue. Rosenfeld and others '~ showed that 5.25% sodium hypochlorite was more effective than saline solution and that it exerted a nonspecific, surface-acting solvent action on intact vital pulp tissue. T h e solvent action was limited by a small lumen and was most effective in the middle and occlusal thirds of the pulp. The purpose of this study was to determine the solvent effect of various dilutions of sodium hypochlorite solution on vital and necrotic tissue. METHODS
AND
MATERIALS
Clorox, a household bleaching agent with 5.25% available chlorine, was diluted with distilled water. Solutions of sodium hypochlorite in 10 ml quantition were m a d e accord-
ing to the following formula: Percentage X volume = percentage X volume, for example, 5.25 X X = 5 x 10, X = 9.52. A 10 ml solution that contains a concentration of 5% available chlorine would consist of 9.5 ml (5.25% sodium hypochlorite and 0.5 ml distilled water). Similarly, 3% concentration = 5.7 ml (5.25% NaOC1) + 4.3 ml distilled water, 1% concentration = 1.9 ml (5.25% NaOCI) + 8.1 ml distilled water. Vital pulp was obtained from bovine teeth. The teeth had been removed from the jaws and were frozen at - 2 0 C. The pulp tissue was removed from eight molars, washed with distilled water, and centrifuged at 8,000 rpm for 15 minutes. The supernatant fluid was removed, and the remaining pellet lyophilized overnight. T h e lyophilized vital pulp was then a d d e d to preweigbed vortex glass test tubes and relyophilized. The combined tube and sample weight was then determined. All weights were obtained using a Mettler H 20 balance. Average tissue weight was 4 rag. All test tubes were carefully marked and sealed, when not in use, and stored in a deep freeze at - 2 0 C. Experiments were conducted at room temperature. Before any experimental procedure was carried out, the pulp tissue was rehydrated with 0.21 ml of distilled water. T h e test tubes were then centrifuged at 3,015 rpm in a Dynac centrifuge for 15 minutes. The supernatant was carefully removed with a pipette. T h e shape of the base of the vortex tube generally allowed for the collection of a solid pellet of tissue after centrifuging. T h e incubation of the tooth pulp with the Glorox was carried out as follows: T h e a m o u n t of N a O C I solution added to the pulp tissue was
controlled so that the concentration of bleach to tissues was constant. Generally 0.5 ml of bleach per 5 m g of tissue was added. Bleach was added to the tissue after which each tube was placed on a Vortex genie vibrator for 5 to 10 seconds. The tube was then incubated for two to ten minutes at 25 C. One-tenth milliliter of 0.1% sodium thiosulfate was added after each specific time period to inactivate the chlorine action, a n d the test tube was placed in ice. T h e samples were centrifuged and the supernatant removed, and then washed twice with distilled water. The samples were again centrifuged for 5 minutes; the supernatant removed, and the pellet washed with water twice. T h e test tubes and contents were then lyophilized a n d reweighed. A similar method was followed f o r the necrotic pulp tissue. The only difference was that the pulp tissue was allowed to bench-stand for one day at 20 C before it was exposed to NaOC1. T h e autolyzed pulp was then relyophilized. T h e texture of the tissue was very fibrous. RESULTS Fetal bovine pulp was prepared as described in the aforementioned text, and then incubated with 0%, 1%, 3%, or 5% NaOC1 for 2, 5, or 10 minutes at room temperature. T h e a m o u n t of pulp per milliliter of N a O C I was standardized at 10 m g / m l for the results shown in Figure 1. Approximately 5 mg aliquots of pulp were used for each time point. The percentage of vital pulp digested by this procedure at 2, 5, and 10 minutes is shown in Figure 1. It can be seen that distilled water had little effect in dissolving vital pulp. Less than 10% of the pulp was lost after 10 minutes. 467
Table 9 Dissolution of vital tooth pulp by 5% NaOCl.
Time of exposure (min)
mg pulp/ml NaOC1
% dissolved
10 10 10
10 20 50
73 74 60
NaOC1 at 1% had a slightly greater ability to dissolve pulp, dissolving about 37% within two minutes of exposure. Longer incubation times showed no increase in solution. NaOC1 at 3 and 5% each-dissolved 70% of the pulp within two minutes, and there was no increase in the percentage dissolved in ten minutes. Necrotic pulp tissue was also tested to determine the percentage of pulp material that would be dissolved by NaOC1. The vital pulp was made necrotic by letting it benchstand for 24 hours at 20 C. The same experiment was then performed on the necrotic pulp that was performed on the vital pulp. Distilled water dissolved a larger percentage of the necrotic pulp than vital. The results in Figure 2 shows that almost 30% of the pulp was susceptible to dissolution by distilled water. The I, 3 and 5% NaOC1 solutions showed almost equal effect on the necrotic pulp. Seventy to 75% of the pulp was dissolved within two minutes, and 85 to 90% of the pulp in five minutes. 468
The amount of tissue dissolved did not change significantly between five to 10 minutes. To determine if the total amount of NaOC1 was an important factor in the solvent effect, vital pulp in concentrations of 10, 20 and 50 mg per ml of 5% NaOC1 was incubated for ten minutes. The results are shown in the Table. It can be seen that the relative amount of pulp had little effect at a concentration of less than 50 mg pulp/ml of NaOC1. However, at a concentration of 50 mg pulp/mI of NaOC1 only 60% of the pulp was dissolved. The reason for this decrease appeared to be because the NaOC1 that could not completely wet the large amount of pulp. DISCUSSION This study confirms that 3% or greater of NaOCI is a more effective tissue solvent than 1%, when tested under the aforementioned conditions. Distilled water is shown to be an ineffective solvent of vital tooth
pulp. More than 70% of necrotic pulp tissue was dissolved in all concentrations of NaOC1 tested. Distilled water dissolved more than 30% of the necrotic pulp. These results seem to confirm the work of Sp~ngberg ~~ who suggested that 0.5% NaOC1 was an effective necrotic tissue solvent. However, our results refute those of Baker and others, 12 who stated that normal saline solution was as effective as 1% NaOC1 as an irrigant. Although we did not test saline solution as such, it would be expected that saline solution would behave much the same as H~O. Trepangier and others 16 have reported that 2.5% NaOC1 is as effective a solvent for root canal irrigation as 5%. Again, although we did not test 2.5%, our results show that 3% and 5% NaOC1 have about equal effect in dissolving the vital pulp, and it would be expected that 2.5% would have about the same degree of effectiveness. Our results also show that necrotic pulp would certainly be dissolved equally well by 2.5 or 5% NaOC1. One can speculate that the greater dissolution of necrotic tissue after exposure to water or 1% NaOGl was due to the necrotizing process, which dissolved some of the pulp. The surface area of the tissue specimens exposed to the NaOC1 is important in determining the degree of dissolution. In fact, in several preliminary experiments (results not shown), it was found that when the pulp was not pulverized and large pieces of pulp were used, it made a significant difference in the percentage of pulp that was dissolved. It was also thought that the total amount of NaOC1, and not the percentage, was the important variable in determining how much NaOC1 is necessary to completely dissolve the tooth pulp. This is supported by the data in the
JOURNAL OF ENDODONTICS I VOL 7, NO 10, OCTOBER 1981
T a b l e . W h e n the a m o u n t o f p u l p / m l of N a O C I r e a c h e d 50 m g there was a slight d r o p - o f f in the p e r c e n t a g e of pulp dissolved, H o w e v e r , at this point, t h e r e was so m u c h p u l p in p r o p o r t i o n to the s o l v e n t t h a t the N a O C 1 c o u l d not w e t it c o m p l e t e l y . These are not t h e c o n d i t i o n s of t h e e x p e r i m e n t s d e s c r i b e d in the text of the p a p e r ; N a O C 1 (5%) was p r e s e n t in excess r e l a t i v e to t h e p u l p . A l t h o u g h there was a n excess of N a O C I to p u l p w h e n t h e root c a n a l was irrigated, it was difficult to evaluate the i m p o r t a n c e o f these results in r e g a r d to the c o n c e n t r a t i o n of N a O C 1 t h a t s h o u l d be used in the canal. I f t h e clinical c o n d i t i o n s w e r e c o m p a r a b l e in t h e root c a n a l to the experimental conditions, a minimum of 3% N a O C 1 for five m i n u t e s w o u l d be r e c o m m e n d e d . H o w e v e r , c o n d i tions in vitro are n o t e x a c t l y like those in the root canal. It is n o t certain if p u l v e r i z a t i o n a n d lyophilization s i g n i f i c a n t l y c h a n g e the p u l p ' s innate susceptibility to N a O C I , alt h o u g h it is unlikely. H o w e v e r , the process c e r t a i n l y increases the surface a r e a a n d m o s t p r o b a b l y facilitates dissolution by i n c r e a s i n g access of the N a O C 1 to the p u l p . A n d it is not k n o w n w h a t i n h i b i t o r s o f N a O C 1 may be present in t h e root c a n a l , which were not p r e s e n t in vitro. Necrotic canals m o s t likely c o n t a i n material r e s u l t i n g f r o m n e c r o t i z i n g agents a n d the b o d y ' s response to them. N e c r o t i c p u l p p r o d u c e d in vitro c o n t a i n s no s u b s t a n c e s t h a t are the result of an i m m u n e r e a c t i o n . T h e injurious effects o f s o d i u m h y p o c h l o r i t e s o l u t i o n o n vital p u l p must be c o n s i d e r e d in r e g a r d to coneentrations. Little o r no d e b r i d e m e n t of vital tissue was f o u n d by G r e y 19 after i n s t r u m e n t i n g v i t a l teeth w i t h saline or s o d i u m h y p o c h l o r i t e solu-
tions. H e c o n c l u d e d t h a t t h e vascul a r i t y resisted the a c t i o n o f N a O C I . M c C o m b a n d S m i t h , ~:~ u s i n g a scann i n g e l e c t r o n m i c r o s c o p e , c a m e to the s a m e conclusion. H o w e v e r , Rosenfeld ~ s h o w e d t h a t t h e r e was a n o n s p e c i f i c n o n c o a g u l a t i n g effect on vital, y o u n g h e a l t h y h u m a n p u l p tissue a n d t h a t the solvent effect was m e r e l y restricted by t h e size o f the l u m e n a n d not by t h e v i a b i l i t y or v a s c u l a r i t y of the tissue.
CONCLUSION A n in vitro i n v e s t i g a t i o n was cond u c t e d to d e t e r m i n e the effect of d i l u t i o n on the solvent p o t e n t i a l of sodium h y p o c h l o r i t e s o l u t i o n at v a r y i n g t i m e intervals. T h e d e c i s i o n r e g a r d i n g w h e t h e r to use 3 o r 5% N a O C l d e p e n d s on t h e l e n g t h of t i m e t h e N a O C I will be in c o n t a c t w i t h the p u l p a n d w h e t h e r h i g h e r concentrations of NaOC1 cause g r e a t e r tissue d a m a g e t h a n l o w e r levels o f N a O C 1 . Dr. Gordon was a graduate student in endodontics, School of Dental Medicine, University of Pennsylvania, when this work was completed, tle is a clinical assistant professor of endodontics, University of Texas Dental School, San Antonio, Tex. Ms. Damato is a research assistant, department of histology, School of Dental Medicine, University of Pennsylvania, Philadelphia. Dr. Christner is assistant professor of histology, Sch(• of Dental Medicine, University of Pennsylvania, Philadelphia, 19104. Requests for reprints should be directed to I)r. Christner. References 1. Penick, E.G., and Osetek, E.M. Intracahal drugs and chemicals in endodontics. Dent Glin N Am 14:743, 1970. 2. Dakin, H.D. On the use of certain antiseptic substances in the treatment of infected wounds. Brit Med J 2:318, 1915. 3. Austin, J.H., and Taylor, H.D. Behaviour of hypochlorite solution in contact with necrotic and normal tissues in vivo. J Exp Med 27:624, 1918. 4. Walker, A. A definite and dependable
therapy for pulpless teeth. JADA 23:1418, 1936. 5. Grossman, L.I., and Meiman, B. Solution of pulp tissue by chemical agents. ,JAI)A 28:223, 1941. 6. Grossman, L.I. Conservative treatment of pulpless teeth..JADA 28:1244, 1941. 7. Lewis, P. Sodium hypnchlorite root canal therapy. J Fla Dent Soc 24:10, 1954. 8. Senia, E.S.: Marshall, F.J.; and Rosen, S, The solvent action of sodium hypochlorite on pulp tissue of extracted tissue. Oral Surg 31:96, 1971. 9. Lamers, A.C., and others. Practical use of sodium hypochlorite in endodontics. Ned Tidskrif 79:142, 1972. 10. Spangberg, I,.; Engstrom, D.; and Langeland. K. Biological effects of dental materials. Tnxicity and antimicrobial effects of endodontic antiseptics in vitro. Oral Surg 36:856, 1973. 11. Trowbridge, It.O. Discussion: cellular reaction to intracanal medicaments. In Transactions of the Fifth International Confi'.rence in Endodontics. Philadelphia, University of Pennsylvania, 1973, p 124. 12. Baker, N.A., and others. Scanning electron microscope study of the efficacy of various irrigating solutions. J Endod 7:127, 1975. 13. McComb, D., and Smith, D.C. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1:238, 1975. 14. Rutberg, M.; Sp~.ngberg, E.; and Spangberg, L. Evaluation of enhanced vascular permeability of endodontic medicaments in vivo. J Endod 3:347, 1977. 15. Svec, T.A., and Harrison, J.W. Chemomechanical removal of pulpal and dentinal debris with sodium hypochlorite and hydrogen peroxide vs normal saline solution. J Endod 3:49, 1977. 16. Trepagnier, C.M.; Madden, R.M.; and Lazzari, E.P. Quantitative study of sndium hypochlorite as an in vitro endodontic irrigant. J Endod 3:194, 1977. 17. ltand, R.E.; Smith, M.L.; and Harrison, J.W. Analysis of the effect of dilution on the necrotic tissue dissolution of property of sodium hypochlorite..J Endod 4:60, 1978. 18. Rosenfeld, E.F.; James, G.A.; and Burch, B.S. Vital pulp response to sodium hypochlorite. J Endod 4:140, 1978. 19. Grey, G.C. The capabilities of sodium hypochlorite to digest organic debris from root canals with emphasis on accessory canals. Thesis, Boston University. 1970. "
469
Solvent effect of various dilutions of sodium hypochlorite on vital and necrotic tissue Terence M. Gordon, DDS; Denise Damato, AS; and Paul Christner, PhD
Vital a n d necrotic b o v i n e t o o t h p u l p were e x p o s e d to 0, 1, 3, a n d 5% N a O C I for two to ten m i n u t e s . N a O C I (0%) h a d no dissolving p o w e r o n vital pulp, and small effect o n necrotic pulp. H o w e v e r , 3 a n d 5% N a O C 1 were e q u a l l y effective in dissolving a b o u t three f o u r t h s of the vital p u l p after t w o m i n u t e s ' exposure. N a O C 1 at 1, 3 a n d 5% were equally effective in dissolving 90% of the necrotic pulp after five m i n u t e s ' exposure.
Sodium hypochlorite has been used for many years as an adjunct to thorough mechanical instrumentation. A strongly alkaline chlorineliberating solution, its germicidal activity is related to the formation of hypochlorous acid on release of chlorine gas from solution.' Dakin-" reported the use of 0.5% of sodium hypochlorite solution to irrigate wounds during World War 1. Austin and Taylor 3 showed the solvent action of sodium hypochlorite (Dakin's solution) on nonvital tissue, noticing that the solution was only mildly inflammatory to normal tissue. Double strength chlorinated soda solution was first recommended empirically for use as a canal irrigant by Walker ~ in 1936. In an experimental study, Grossman and Meiman 5 in 1941 found double strength chlorinated soda solution (3% NaOCI) a most effective solvent, dissolving pulp tissue within 20 minutes to two hours. In the same year Grossman ~ recommended irrigation with hydrogen peroxide (3%) and sodium hypochlorite solution
466
(0.5%). The use of Clorox as a source of chlorinated soda or sodium hypochlorite was introduced by Lewis~ in 1954. In 1971, Senia and others s concluded that 5.25% sodium hypochlorite (Clorox) was not an effective solvent in canals with small diameters. The sodium hypochlorite solution was not used in conjunction with normal clinical procedures, but the solution was allowed to remain in the tooth for a specific time. In 1972, Lamers and others" showed that non-necrotic tissue fixed by paraformaldehyde dissolved more slowly in sodium hypochlorite solution. They also showed that 1% is less irritating and almost as effective as 5% sodium hypochlorite solution. Sp~mgberg and others'" in 1973 suggested the use of 0.5% sodium hypochlorite solution to minimize toxicity and maintain bacteriostatic effectiveness. However, at this dilution, the solvent effect was significantly diminished and was effective only on necrotic tissue. The cytotoxicity studies were based on results using Hela and I, cells. Trowbridge 1~
pointed out that care should be taken in interpreting these cytotoxic results. Baker and associates '~ evaluated various irrigants for their effectiveness in removing tooth canal debris. They found no difference between normal saline solution and 1% sodium hypochlorite solution. Citing the Sp~ngberg study, they recommended the use of saline solution as an irrigant because of its lack of toxicity. McComb and Smith 1'~used a scanning electron microscope and reported that virtually no debris was seen throughout the entire canal after 6% sodium hypochlorite solution was used with instrumentation. The 1% solution was not as effective. In pilot studies using 5% sodium hypochlorite solution, Rutberg and others" found extensive tissue damage, and the affected tissue was necrotised and dissolved. Svec and Harrison '~ showed that a combination of 5.25% sodium hypochlorite solution and 3% hydrogen peroxide was significantly more effective in cleaning the canal system at 1- and 3-mm apical levels. At the 5-mm level, normal saline solution was equally effective as an irrigant. Trepagnier and others TM reported a quantitative evaluation of different dilutions of sodium hypochlorite solution. A determination of the dissolution and removal of pulpal and dentinal debris from the canal was
JOURNAL OF ENDODONTICS I VOL 7, NO 10, OCTOBER 1981
measured by analyzing the hydroxyproline content of the solution that resulted after irrigation. Pulp tissue consists of 15% of collagen, containing approximately 13% hydroxyprolinc. The results indicated that sodium hypochlorite solution is a powerful solvent whose action starts immediately and continues for at least an hour. Dilution to 2.5% does not affect its solvent action appreciably, but a modified Dakin's solution has little solvent action. They, therefore, suggested the use of the diluted solution, that is, 2.5%. H a n d and others '7 evaluated the effect of diluting sodium hypochlorite solution on its solvent action. They exposed necrotic tissue to various concentrations of sodium hypochlorite solution, and the percentage of tissue weight changes was determined. Statistical analysis indicated that dilution of 5.25% sodium hypochlorite resulted in a significant decrease in its ability to dissolve necrotic tissue. Rosenfeld and others '~ showed that 5.25% sodium hypochlorite was more effective than saline solution and that it exerted a nonspecific, surface-acting solvent action on intact vital pulp tissue. T h e solvent action was limited by a small lumen and was most effective in the middle and occlusal thirds of the pulp. The purpose of this study was to determine the solvent effect of various dilutions of sodium hypochlorite solution on vital and necrotic tissue. METHODS
AND
MATERIALS
Clorox, a household bleaching agent with 5.25% available chlorine, was diluted with distilled water. Solutions of sodium hypochlorite in 10 ml quantition were m a d e accord-
ing to the following formula: Percentage X volume = percentage X volume, for example, 5.25 X X = 5 x 10, X = 9.52. A 10 ml solution that contains a concentration of 5% available chlorine would consist of 9.5 ml (5.25% sodium hypochlorite and 0.5 ml distilled water). Similarly, 3% concentration = 5.7 ml (5.25% NaOC1) + 4.3 ml distilled water, 1% concentration = 1.9 ml (5.25% NaOCI) + 8.1 ml distilled water. Vital pulp was obtained from bovine teeth. The teeth had been removed from the jaws and were frozen at - 2 0 C. The pulp tissue was removed from eight molars, washed with distilled water, and centrifuged at 8,000 rpm for 15 minutes. The supernatant fluid was removed, and the remaining pellet lyophilized overnight. T h e lyophilized vital pulp was then a d d e d to preweigbed vortex glass test tubes and relyophilized. The combined tube and sample weight was then determined. All weights were obtained using a Mettler H 20 balance. Average tissue weight was 4 rag. All test tubes were carefully marked and sealed, when not in use, and stored in a deep freeze at - 2 0 C. Experiments were conducted at room temperature. Before any experimental procedure was carried out, the pulp tissue was rehydrated with 0.21 ml of distilled water. T h e test tubes were then centrifuged at 3,015 rpm in a Dynac centrifuge for 15 minutes. The supernatant was carefully removed with a pipette. T h e shape of the base of the vortex tube generally allowed for the collection of a solid pellet of tissue after centrifuging. T h e incubation of the tooth pulp with the Glorox was carried out as follows: T h e a m o u n t of N a O C I solution added to the pulp tissue was
controlled so that the concentration of bleach to tissues was constant. Generally 0.5 ml of bleach per 5 m g of tissue was added. Bleach was added to the tissue after which each tube was placed on a Vortex genie vibrator for 5 to 10 seconds. The tube was then incubated for two to ten minutes at 25 C. One-tenth milliliter of 0.1% sodium thiosulfate was added after each specific time period to inactivate the chlorine action, a n d the test tube was placed in ice. T h e samples were centrifuged and the supernatant removed, and then washed twice with distilled water. The samples were again centrifuged for 5 minutes; the supernatant removed, and the pellet washed with water twice. T h e test tubes and contents were then lyophilized a n d reweighed. A similar method was followed f o r the necrotic pulp tissue. The only difference was that the pulp tissue was allowed to bench-stand for one day at 20 C before it was exposed to NaOC1. T h e autolyzed pulp was then relyophilized. T h e texture of the tissue was very fibrous. RESULTS Fetal bovine pulp was prepared as described in the aforementioned text, and then incubated with 0%, 1%, 3%, or 5% NaOC1 for 2, 5, or 10 minutes at room temperature. T h e a m o u n t of pulp per milliliter of N a O C I was standardized at 10 m g / m l for the results shown in Figure 1. Approximately 5 mg aliquots of pulp were used for each time point. The percentage of vital pulp digested by this procedure at 2, 5, and 10 minutes is shown in Figure 1. It can be seen that distilled water had little effect in dissolving vital pulp. Less than 10% of the pulp was lost after 10 minutes. 467
Table 9 Dissolution of vital tooth pulp by 5% NaOCl.
Time of exposure (min)
mg pulp/ml NaOC1
% dissolved
10 10 10
10 20 50
73 74 60
NaOC1 at 1% had a slightly greater ability to dissolve pulp, dissolving about 37% within two minutes of exposure. Longer incubation times showed no increase in solution. NaOC1 at 3 and 5% each-dissolved 70% of the pulp within two minutes, and there was no increase in the percentage dissolved in ten minutes. Necrotic pulp tissue was also tested to determine the percentage of pulp material that would be dissolved by NaOC1. The vital pulp was made necrotic by letting it benchstand for 24 hours at 20 C. The same experiment was then performed on the necrotic pulp that was performed on the vital pulp. Distilled water dissolved a larger percentage of the necrotic pulp than vital. The results in Figure 2 shows that almost 30% of the pulp was susceptible to dissolution by distilled water. The I, 3 and 5% NaOC1 solutions showed almost equal effect on the necrotic pulp. Seventy to 75% of the pulp was dissolved within two minutes, and 85 to 90% of the pulp in five minutes. 468
The amount of tissue dissolved did not change significantly between five to 10 minutes. To determine if the total amount of NaOC1 was an important factor in the solvent effect, vital pulp in concentrations of 10, 20 and 50 mg per ml of 5% NaOC1 was incubated for ten minutes. The results are shown in the Table. It can be seen that the relative amount of pulp had little effect at a concentration of less than 50 mg pulp/ml of NaOC1. However, at a concentration of 50 mg pulp/mI of NaOC1 only 60% of the pulp was dissolved. The reason for this decrease appeared to be because the NaOC1 that could not completely wet the large amount of pulp. DISCUSSION This study confirms that 3% or greater of NaOCI is a more effective tissue solvent than 1%, when tested under the aforementioned conditions. Distilled water is shown to be an ineffective solvent of vital tooth
pulp. More than 70% of necrotic pulp tissue was dissolved in all concentrations of NaOC1 tested. Distilled water dissolved more than 30% of the necrotic pulp. These results seem to confirm the work of Sp~ngberg ~~ who suggested that 0.5% NaOC1 was an effective necrotic tissue solvent. However, our results refute those of Baker and others, 12 who stated that normal saline solution was as effective as 1% NaOC1 as an irrigant. Although we did not test saline solution as such, it would be expected that saline solution would behave much the same as H~O. Trepangier and others 16 have reported that 2.5% NaOC1 is as effective a solvent for root canal irrigation as 5%. Again, although we did not test 2.5%, our results show that 3% and 5% NaOC1 have about equal effect in dissolving the vital pulp, and it would be expected that 2.5% would have about the same degree of effectiveness. Our results also show that necrotic pulp would certainly be dissolved equally well by 2.5 or 5% NaOC1. One can speculate that the greater dissolution of necrotic tissue after exposure to water or 1% NaOGl was due to the necrotizing process, which dissolved some of the pulp. The surface area of the tissue specimens exposed to the NaOC1 is important in determining the degree of dissolution. In fact, in several preliminary experiments (results not shown), it was found that when the pulp was not pulverized and large pieces of pulp were used, it made a significant difference in the percentage of pulp that was dissolved. It was also thought that the total amount of NaOC1, and not the percentage, was the important variable in determining how much NaOC1 is necessary to completely dissolve the tooth pulp. This is supported by the data in the
JOURNAL OF ENDODONTICS I VOL 7, NO 10, OCTOBER 1981
T a b l e . W h e n the a m o u n t o f p u l p / m l of N a O C I r e a c h e d 50 m g there was a slight d r o p - o f f in the p e r c e n t a g e of pulp dissolved, H o w e v e r , at this point, t h e r e was so m u c h p u l p in p r o p o r t i o n to the s o l v e n t t h a t the N a O C 1 c o u l d not w e t it c o m p l e t e l y . These are not t h e c o n d i t i o n s of t h e e x p e r i m e n t s d e s c r i b e d in the text of the p a p e r ; N a O C 1 (5%) was p r e s e n t in excess r e l a t i v e to t h e p u l p . A l t h o u g h there was a n excess of N a O C I to p u l p w h e n t h e root c a n a l was irrigated, it was difficult to evaluate the i m p o r t a n c e o f these results in r e g a r d to the c o n c e n t r a t i o n of N a O C 1 t h a t s h o u l d be used in the canal. I f t h e clinical c o n d i t i o n s w e r e c o m p a r a b l e in t h e root c a n a l to the experimental conditions, a minimum of 3% N a O C 1 for five m i n u t e s w o u l d be r e c o m m e n d e d . H o w e v e r , c o n d i tions in vitro are n o t e x a c t l y like those in the root canal. It is n o t certain if p u l v e r i z a t i o n a n d lyophilization s i g n i f i c a n t l y c h a n g e the p u l p ' s innate susceptibility to N a O C I , alt h o u g h it is unlikely. H o w e v e r , the process c e r t a i n l y increases the surface a r e a a n d m o s t p r o b a b l y facilitates dissolution by i n c r e a s i n g access of the N a O C 1 to the p u l p . A n d it is not k n o w n w h a t i n h i b i t o r s o f N a O C 1 may be present in t h e root c a n a l , which were not p r e s e n t in vitro. Necrotic canals m o s t likely c o n t a i n material r e s u l t i n g f r o m n e c r o t i z i n g agents a n d the b o d y ' s response to them. N e c r o t i c p u l p p r o d u c e d in vitro c o n t a i n s no s u b s t a n c e s t h a t are the result of an i m m u n e r e a c t i o n . T h e injurious effects o f s o d i u m h y p o c h l o r i t e s o l u t i o n o n vital p u l p must be c o n s i d e r e d in r e g a r d to coneentrations. Little o r no d e b r i d e m e n t of vital tissue was f o u n d by G r e y 19 after i n s t r u m e n t i n g v i t a l teeth w i t h saline or s o d i u m h y p o c h l o r i t e solu-
tions. H e c o n c l u d e d t h a t t h e vascul a r i t y resisted the a c t i o n o f N a O C I . M c C o m b a n d S m i t h , ~:~ u s i n g a scann i n g e l e c t r o n m i c r o s c o p e , c a m e to the s a m e conclusion. H o w e v e r , Rosenfeld ~ s h o w e d t h a t t h e r e was a n o n s p e c i f i c n o n c o a g u l a t i n g effect on vital, y o u n g h e a l t h y h u m a n p u l p tissue a n d t h a t the solvent effect was m e r e l y restricted by t h e size o f the l u m e n a n d not by t h e v i a b i l i t y or v a s c u l a r i t y of the tissue.
CONCLUSION A n in vitro i n v e s t i g a t i o n was cond u c t e d to d e t e r m i n e the effect of d i l u t i o n on the solvent p o t e n t i a l of sodium h y p o c h l o r i t e s o l u t i o n at v a r y i n g t i m e intervals. T h e d e c i s i o n r e g a r d i n g w h e t h e r to use 3 o r 5% N a O C l d e p e n d s on t h e l e n g t h of t i m e t h e N a O C I will be in c o n t a c t w i t h the p u l p a n d w h e t h e r h i g h e r concentrations of NaOC1 cause g r e a t e r tissue d a m a g e t h a n l o w e r levels o f N a O C 1 . Dr. Gordon was a graduate student in endodontics, School of Dental Medicine, University of Pennsylvania, when this work was completed, tle is a clinical assistant professor of endodontics, University of Texas Dental School, San Antonio, Tex. Ms. Damato is a research assistant, department of histology, School of Dental Medicine, University of Pennsylvania, Philadelphia. Dr. Christner is assistant professor of histology, Sch(• of Dental Medicine, University of Pennsylvania, Philadelphia, 19104. Requests for reprints should be directed to I)r. Christner. References 1. Penick, E.G., and Osetek, E.M. Intracahal drugs and chemicals in endodontics. Dent Glin N Am 14:743, 1970. 2. Dakin, H.D. On the use of certain antiseptic substances in the treatment of infected wounds. Brit Med J 2:318, 1915. 3. Austin, J.H., and Taylor, H.D. Behaviour of hypochlorite solution in contact with necrotic and normal tissues in vivo. J Exp Med 27:624, 1918. 4. Walker, A. A definite and dependable
therapy for pulpless teeth. JADA 23:1418, 1936. 5. Grossman, L.I., and Meiman, B. Solution of pulp tissue by chemical agents. ,JAI)A 28:223, 1941. 6. Grossman, L.I. Conservative treatment of pulpless teeth..JADA 28:1244, 1941. 7. Lewis, P. Sodium hypnchlorite root canal therapy. J Fla Dent Soc 24:10, 1954. 8. Senia, E.S.: Marshall, F.J.; and Rosen, S, The solvent action of sodium hypochlorite on pulp tissue of extracted tissue. Oral Surg 31:96, 1971. 9. Lamers, A.C., and others. Practical use of sodium hypochlorite in endodontics. Ned Tidskrif 79:142, 1972. 10. Spangberg, I,.; Engstrom, D.; and Langeland. K. Biological effects of dental materials. Tnxicity and antimicrobial effects of endodontic antiseptics in vitro. Oral Surg 36:856, 1973. 11. Trowbridge, It.O. Discussion: cellular reaction to intracanal medicaments. In Transactions of the Fifth International Confi'.rence in Endodontics. Philadelphia, University of Pennsylvania, 1973, p 124. 12. Baker, N.A., and others. Scanning electron microscope study of the efficacy of various irrigating solutions. J Endod 7:127, 1975. 13. McComb, D., and Smith, D.C. A preliminary scanning electron microscopic study of root canals after endodontic procedures. J Endod 1:238, 1975. 14. Rutberg, M.; Sp~.ngberg, E.; and Spangberg, L. Evaluation of enhanced vascular permeability of endodontic medicaments in vivo. J Endod 3:347, 1977. 15. Svec, T.A., and Harrison, J.W. Chemomechanical removal of pulpal and dentinal debris with sodium hypochlorite and hydrogen peroxide vs normal saline solution. J Endod 3:49, 1977. 16. Trepagnier, C.M.; Madden, R.M.; and Lazzari, E.P. Quantitative study of sndium hypochlorite as an in vitro endodontic irrigant. J Endod 3:194, 1977. 17. ltand, R.E.; Smith, M.L.; and Harrison, J.W. Analysis of the effect of dilution on the necrotic tissue dissolution of property of sodium hypochlorite..J Endod 4:60, 1978. 18. Rosenfeld, E.F.; James, G.A.; and Burch, B.S. Vital pulp response to sodium hypochlorite. J Endod 4:140, 1978. 19. Grey, G.C. The capabilities of sodium hypochlorite to digest organic debris from root canals with emphasis on accessory canals. Thesis, Boston University. 1970. "
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