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Effect of protein and sodium hypochlorite on endodontic instruments
JOURNAL OF ENDODONTICS I VOL 2, NO 11, NOVEMBER 1976
E f f e c t of p r o t e i n a n d s o d i u m h y p o c h l o r i t e on endodontic instruments Marvin A. Eichner, DDS; David M. Schoen, DMD; Melvin Goldman, DDS~ and Joseph H. Kronman DDS, Phi), Boston
W h e n c a r b o n steel e n d o d o n t i c instruments w e r e i m m e r s e d in 1% s o d i u m h y p o c h l o r i t e for ten minutes, p h o t o q r a p h s did not s h o w , o n a one-to-one c o m p a r a t i v e basis, corrosion of the instruments. W i t h 5% Na 9 corrosion w a s present. W h e n purified b o v i n e c o l l a q e n w a s a d d e d to b o t h 1% a n d 5% N a 9 corrosion w a s present. The q r e a t e s t corrosion w a s s e e n w h e n the c o m b i n a t i o n of 5 % Na 9 a n d purified b o v i n e c o l l a q e n w a s used. W h e n stainless steel e n d o d o n t i c instruments w e r e i m m e r s e d in 1% a n d 5 % Na 9 with or w i t h o u t purified b o v i n e c o l l a q e n p r e s e n t in the solution, p h o t o q r a p h s d i d not show, on a one-to-one c o m p a r a t i v e basis, corrosion of the instruments.
Craig and associates 1-s showed the similarity of the physical and mechanical properties of stainless and carbon steel endodontic files and reamers. However, stainless steel instruments had a generally improved resistance to breakage from torquing forces. They concluded ,that the hardness of both carbon and stainless steel instruments used would lead to little difference in their ability to cut dentin. Gutierrez, Gigoux, and Sanhueza 4 used light microscopy to examine a total of 665 new and used carbon and stainless steel reamers and files for "rolling up," "unrolling," "notching," and other signs of wear. They also observed that sodium hypochlorite had a blistering effect on carbon steel, but they did not report the effect of
NaOC1 on stainless steel instruments. They concluded that endodontic reamers should be made from stainless steelA Oliet and Sorin 5 reported on the cutting efficacy of reamers as related to manufacture, design, size, and material. Neither carbon nor stainless steel was shown to be a consistently superior cutting material. Manufacture and design were significant variables for instruments marketed as equivalents. It may be that the working properties of carbon and stainless steel instruments are clinically equivalent. Whatever the physical differences are, they may be masked by the tremendous variation in manufacturing procedures and design. These differences
may be compensated in varying amounts by the clinician. Thus, in terms of selecting suitable endodontic instruments, it would seem difficult to select either carbon or stainless steel endodorttic instruments by working characteristics alone, unless one also includes the reactivity of the metal as a working characteristic. For this reason it was decided to evaluate the reactivity of carbon and stainless steel instruments with NaOC1, a commonly used irrigant. Several pilot studies were conducted before designing this experiment. These preliminary studies suggested that the corrosive activity of NaOC1 was much greater clinically than it was in the test tube. In an attempt to identify what
335
JOURNAL OF ENDODONTICS t VOL 2, NO 11, NOVEMBER 1976
might be responsible for this accelerated corrosion, NaOCI was reacted with endodontic files in the presence of solutions containing purified proteins. These solutions clearly accelerated the corrosion process. After testing several solutions containing purified proteins, a solution of bovine collagen appeared to produce results comparable to those seen clinically. For this reason, a solution of purified bovine collagen was selected as the substrate for the present experiment. The purpose of this investigation was .to determine the relative reactivities of carbon and stainless steel endodontic instruments exposed to various concentrations of NaOC1 in the presence and absence of purified bovine collagen. Materials and Methods Thirty-six stainless steel and 36 carbon steel endodontic instruments,* purchased on the open market, were used. Twelve groups of instruments were studied, with six instruments in each group. There were six carbon steel instrument groups and six stainless steel instrument groups, as follows: group 1, carbon steel instruments in 5% NaOC1; group 2, carbon steel instruments in 5% NaOC1 plus bovine collagen; group 3, carbon steel instrum e n t s in 1% NaOC1; group 4, carbon steel instruments in 1% NaOC1 plus bovine collagen; group 5, stainless steel instruments in 5% NaOC1; group 6, stainless steel instruments in 5% NaOC1 plus bovine collagen; group 7, stainless steel instruments in 1% NaOC1; group 8, stainless steel instruments in 1% NaOC1 plus bovine collagen; group 9, carbon steel instruments in water; group 10, carbon steel instruments in water plus bovine collagen; group 11, stainless steel instruments in water; and group 12, stainless steel instruments in water plus bovine collagen.
336
The time period used in all categories was ten minutes. Cloroxt was used as the source of 5% NaOCI. Clorox diluted in a ratio of 1:4 with deionized water was used as the source of 1% NaOCI. Purified bovine collagen was obtained from a commercial source.:~ The instruments were reacted individually in 4-ml .test tubes. The quantity of bovine collagen was standardized at 30 mg • rng per cube. The fluid level in each test tube was brought to within three eighths of an inch of the mouth of the tube to avoid contact with the instrument handle. To facilitate handling of the instruments, four banks of 12 test tubes were fabricated. After placing the bovine collagen in the appropriate tubes, a polyester mesh was inserted into all tubes to about two thirds of the total tube depth. The mesh was inserted to keep the bovine collagen submerged and to prevent direct contact with the blade of the instrument. The mesh was placed in all tubes to eliminate any variation that the mesh itself might introduce. The instruments were fastened to glass slides by the butts of their handles via a two-sided adhesive tape and arranged on the slides so that when the slides were inverted over the bank of test tubes, the instruments would be suspended within the lumen of their respective tubes. After being exposed for ten minutes, the instruments were immersed in a tank of water for 30 seconds with no agitation. This was done to limit the action of the v~rious solutions and to clean the surfaces of the instruments for photographic purposes. The instruments then were dried for 15 minutes at a temperature of 70 C. After drying, each group was photographed at an image ratio of 1:1. In addition, combined group photographs were made at an image ratio of 1:10.
Results The photographs did not show a visible corrosion of the stainless steel instruments when subjected to any of the experimental conditions, or visible corrosion of the carbon steel instruments in either water, water plus collagen, or 1% NaOC1 without collagen. The photographs did show observable corrosion on carbon steel instruments exposed to 1% NaOC1 plus collagen, 5% NaOC1, and 5% NaOCI plus collagen. The following observations were made: 1% NaOC1 plus collagen produced a light, uniform coating of corrosion on the instrument (Fig 1); 5% NaOC1 plus collagen produced a heavy, uniform coating o f corrosion on the instrument (Fig 2); and 5% NaOC1 produced small amounts of corrosion randomly distributed on the instruments. Many large, unaffected areas were noticed (Fig 3). The amounts of corrosion observed on carbon steel instruments were always greater in the groups that contained collagen than in those that did not.
Discussion Carbon steel instruments corroded in the presence of 5% NaOCI. This corrosion was greatly enhanced by the presence of protein in the form of purified bovine collagen (Figures 4 and 5). With 1% NaOC1 and no collagen, there was no corrosion. Howe v e r , where collagen was added, corrosion was evident. On the other hand, stainless steel instruments did not appear to corrode in any concentration of NaOC1 used, with or without collagen. It was observed that with the amount of collagen held constant, the degree of corrosion on the carbon instruments increased as the concentrations of NaOC1 increased. Additional trials after conclusion of the study reported here showed that the amount
JOURNAL OF ENDODONTICS [ VOL 2, NO 11, NOVEMBER 1976
Fig 1---Carbon steel files after being immersed in 1% NaOCl and purified bovine collagen for ten minutes. Notice uniform, light corrosion on instruments.
Fig 2----Carbon steel files after being immersed in 5% NaOCl and collagen for ten minutes. Notice uniform, heavy corrosion.
Fig 3--Carbon steel files after being immersed in 5% NaOCl without collagen for ten minutes. Notice randomly distributed clumps of corrosion and large areas that appear to be unaffected.
of corrosion increased as the amount of collagen present increased, when the other factors remained constant. Because all root canals contain protein, this may be an important aspect to consider in clinical endodontics. Of course, a solution of purified bovine collagen is not the same solution of proteins that one would encounter in a root canal clinically. Nevertheless, the experimental solution was considered to parallel the clinical situation closely enough for the purposes of this study. The photographic technique used was sufficient to detect a real difference between stainless and carbon steel instruments. The ability of collagen to accelerate corrosion of stainless steel instruments could not be shown by photography. If the effect induced by collagen also applies to stainless steel, a more sensitive means of evaluation than photography would be required. The corrosion products have not been identified. They were submitted for analysis to obtain some sense of their protein content. 6 The Lowry and ninhydrin tests were used. Collectively, these tests sample for tryptophan, tyrosine, and alpha amino groups. The ninhydrin determination is capable of identifying appropriate substrates in quantities as small as 0.1/xmol. As such, these tests represent an extremely sensitive assay for protein. Yet when corrosion products were assayed, no protein was detected. Other known characteristics of these corrosion products are as follows: they are heavier than water; they appear to be insoluble in either water or NaOC1; and they are red brown in color. It is likely that they are oxides of iron, but further tests would be required for confirmation. Considering the friability of the corrosion products, one should be concerned with the deposition of these products in the canals and periapical
337
JOURNAL OF ENDODONTICS [ VOL 2, NO I1, NOVEMBER 1976
the carbon steel instruments were always greater in the groups ,that contained collagen than in those that did not. The greatest corrosion was seen when the combination of 5% NaOCI and collagen was used. Although purified bovine collagen is not the same type of protein encountered in root canals clinically, it was considered an adequate parallel for the purpose of this study.
Fig 4---Carbon steel files after ten minutes exposure to NaOCI without collagen: left file, 1% NaOCl; right file, 5 % NaOCl (orig mag • 10).
*Kerr Manufacturing Co., Romulus, Mich. tClorox Co., Oakland, Calif. ~Sigma Chemical Corp., St. Louis. The authors gratefully acknowledge the assistance of Drs. Charles Habib and Steven Davis for their advice during this study.
Fig 5 - - S a m e as in Figure 4 except collagen present in incubating solution with NaOCl: left file, 1% NaOCI plus collagen; right file, 5 % NaOCl plus collagen.
Drs. Eichner and Schoen are graduate students in endodontics; Dr. Goldman is clinical professor in endodontics and director of postgraduate endodontics, and Dr. Kronman is professor of orthodontics, Tufts University School of Dental Medicine, Boston. Requests for reprints should be directed to: Dr. Melvin Goldman, Tufts University School of Dental Medicine, 136 Harrison Ave, Boston, 02111. References
tissue, as well as their possible biologic consequences. The insolubility and specific gravity of these products would seem to make them difficult to remove. The role that corrosion products may play in endodontic failures is unknown. However, this variable can be minimized with the use of stainless steel instruments.
Summary Thirty-six stainless steel and 36 carbon steel endodontic instruments were immersed for ten minutes in solutions of 1% and 5 % NaOC1, with and without the addition of purified bovine collagen. Water, with and withotlt the
338
collagen, served as a control. The instruments were washed, dried, and then photographed at an image ratio of 1:1. The photographs showed no visible corrosion of the stainless steel instruments under any of the experimental conditions, nor did they show any visible corrosion of the carbon steel instruments when they were immersed in either water, water and collagen, or 1% NaOC1 without collagen. The photographs also showed corrosion on carbon steel instruments immersed in 1% NaOC1 and collagen, 5% NaOCI, and 5% NaOC1 and collagen. The amounts of corrosion on
1. Craig, R.G., and Peyton, F.A. Physical properties of carbon steel root canal files and reamers. Oral Surg 15:213 Feb 1962. 2. Craig, R.G., and Peyton, F.A. Physical properties of stainless steel endodontic files and reamers. Oral Surg 16: 206 Feb 1963. 3. Craig, R.G.; Mcllwain, E.D.; and Peyton, F.A. Bending and torsion properties of endodontic instruments. Oral Surg 25:239 Feb 1968. 4. Gutierrez, J.H.; Gigoux, C.; and Sanhueza, I. Physical and chemical deterioration of endodontic reamers during mechanical preparation. Oral Surg 28:394 Sept 1969. 5. Oliet, S., and Sorin, S.M. Cutting efficiency of endodontic reamers. Oral Surg 36:243 Aug 1973. 6. Blackburn, S. Amino acid determination; methods and techniques. New York, Marcel Dekker, Inc., 1968, p 69.
E f f e c t of p r o t e i n a n d s o d i u m h y p o c h l o r i t e on endodontic instruments Marvin A. Eichner, DDS; David M. Schoen, DMD; Melvin Goldman, DDS~ and Joseph H. Kronman DDS, Phi), Boston
W h e n c a r b o n steel e n d o d o n t i c instruments w e r e i m m e r s e d in 1% s o d i u m h y p o c h l o r i t e for ten minutes, p h o t o q r a p h s did not s h o w , o n a one-to-one c o m p a r a t i v e basis, corrosion of the instruments. W i t h 5% Na 9 corrosion w a s present. W h e n purified b o v i n e c o l l a q e n w a s a d d e d to b o t h 1% a n d 5% N a 9 corrosion w a s present. The q r e a t e s t corrosion w a s s e e n w h e n the c o m b i n a t i o n of 5 % Na 9 a n d purified b o v i n e c o l l a q e n w a s used. W h e n stainless steel e n d o d o n t i c instruments w e r e i m m e r s e d in 1% a n d 5 % Na 9 with or w i t h o u t purified b o v i n e c o l l a q e n p r e s e n t in the solution, p h o t o q r a p h s d i d not show, on a one-to-one c o m p a r a t i v e basis, corrosion of the instruments.
Craig and associates 1-s showed the similarity of the physical and mechanical properties of stainless and carbon steel endodontic files and reamers. However, stainless steel instruments had a generally improved resistance to breakage from torquing forces. They concluded ,that the hardness of both carbon and stainless steel instruments used would lead to little difference in their ability to cut dentin. Gutierrez, Gigoux, and Sanhueza 4 used light microscopy to examine a total of 665 new and used carbon and stainless steel reamers and files for "rolling up," "unrolling," "notching," and other signs of wear. They also observed that sodium hypochlorite had a blistering effect on carbon steel, but they did not report the effect of
NaOC1 on stainless steel instruments. They concluded that endodontic reamers should be made from stainless steelA Oliet and Sorin 5 reported on the cutting efficacy of reamers as related to manufacture, design, size, and material. Neither carbon nor stainless steel was shown to be a consistently superior cutting material. Manufacture and design were significant variables for instruments marketed as equivalents. It may be that the working properties of carbon and stainless steel instruments are clinically equivalent. Whatever the physical differences are, they may be masked by the tremendous variation in manufacturing procedures and design. These differences
may be compensated in varying amounts by the clinician. Thus, in terms of selecting suitable endodontic instruments, it would seem difficult to select either carbon or stainless steel endodorttic instruments by working characteristics alone, unless one also includes the reactivity of the metal as a working characteristic. For this reason it was decided to evaluate the reactivity of carbon and stainless steel instruments with NaOC1, a commonly used irrigant. Several pilot studies were conducted before designing this experiment. These preliminary studies suggested that the corrosive activity of NaOC1 was much greater clinically than it was in the test tube. In an attempt to identify what
335
JOURNAL OF ENDODONTICS t VOL 2, NO 11, NOVEMBER 1976
might be responsible for this accelerated corrosion, NaOCI was reacted with endodontic files in the presence of solutions containing purified proteins. These solutions clearly accelerated the corrosion process. After testing several solutions containing purified proteins, a solution of bovine collagen appeared to produce results comparable to those seen clinically. For this reason, a solution of purified bovine collagen was selected as the substrate for the present experiment. The purpose of this investigation was .to determine the relative reactivities of carbon and stainless steel endodontic instruments exposed to various concentrations of NaOC1 in the presence and absence of purified bovine collagen. Materials and Methods Thirty-six stainless steel and 36 carbon steel endodontic instruments,* purchased on the open market, were used. Twelve groups of instruments were studied, with six instruments in each group. There were six carbon steel instrument groups and six stainless steel instrument groups, as follows: group 1, carbon steel instruments in 5% NaOC1; group 2, carbon steel instruments in 5% NaOC1 plus bovine collagen; group 3, carbon steel instrum e n t s in 1% NaOC1; group 4, carbon steel instruments in 1% NaOC1 plus bovine collagen; group 5, stainless steel instruments in 5% NaOC1; group 6, stainless steel instruments in 5% NaOC1 plus bovine collagen; group 7, stainless steel instruments in 1% NaOC1; group 8, stainless steel instruments in 1% NaOC1 plus bovine collagen; group 9, carbon steel instruments in water; group 10, carbon steel instruments in water plus bovine collagen; group 11, stainless steel instruments in water; and group 12, stainless steel instruments in water plus bovine collagen.
336
The time period used in all categories was ten minutes. Cloroxt was used as the source of 5% NaOCI. Clorox diluted in a ratio of 1:4 with deionized water was used as the source of 1% NaOCI. Purified bovine collagen was obtained from a commercial source.:~ The instruments were reacted individually in 4-ml .test tubes. The quantity of bovine collagen was standardized at 30 mg • rng per cube. The fluid level in each test tube was brought to within three eighths of an inch of the mouth of the tube to avoid contact with the instrument handle. To facilitate handling of the instruments, four banks of 12 test tubes were fabricated. After placing the bovine collagen in the appropriate tubes, a polyester mesh was inserted into all tubes to about two thirds of the total tube depth. The mesh was inserted to keep the bovine collagen submerged and to prevent direct contact with the blade of the instrument. The mesh was placed in all tubes to eliminate any variation that the mesh itself might introduce. The instruments were fastened to glass slides by the butts of their handles via a two-sided adhesive tape and arranged on the slides so that when the slides were inverted over the bank of test tubes, the instruments would be suspended within the lumen of their respective tubes. After being exposed for ten minutes, the instruments were immersed in a tank of water for 30 seconds with no agitation. This was done to limit the action of the v~rious solutions and to clean the surfaces of the instruments for photographic purposes. The instruments then were dried for 15 minutes at a temperature of 70 C. After drying, each group was photographed at an image ratio of 1:1. In addition, combined group photographs were made at an image ratio of 1:10.
Results The photographs did not show a visible corrosion of the stainless steel instruments when subjected to any of the experimental conditions, or visible corrosion of the carbon steel instruments in either water, water plus collagen, or 1% NaOC1 without collagen. The photographs did show observable corrosion on carbon steel instruments exposed to 1% NaOC1 plus collagen, 5% NaOC1, and 5% NaOCI plus collagen. The following observations were made: 1% NaOC1 plus collagen produced a light, uniform coating of corrosion on the instrument (Fig 1); 5% NaOC1 plus collagen produced a heavy, uniform coating o f corrosion on the instrument (Fig 2); and 5% NaOC1 produced small amounts of corrosion randomly distributed on the instruments. Many large, unaffected areas were noticed (Fig 3). The amounts of corrosion observed on carbon steel instruments were always greater in the groups that contained collagen than in those that did not.
Discussion Carbon steel instruments corroded in the presence of 5% NaOCI. This corrosion was greatly enhanced by the presence of protein in the form of purified bovine collagen (Figures 4 and 5). With 1% NaOC1 and no collagen, there was no corrosion. Howe v e r , where collagen was added, corrosion was evident. On the other hand, stainless steel instruments did not appear to corrode in any concentration of NaOC1 used, with or without collagen. It was observed that with the amount of collagen held constant, the degree of corrosion on the carbon instruments increased as the concentrations of NaOC1 increased. Additional trials after conclusion of the study reported here showed that the amount
JOURNAL OF ENDODONTICS [ VOL 2, NO 11, NOVEMBER 1976
Fig 1---Carbon steel files after being immersed in 1% NaOCl and purified bovine collagen for ten minutes. Notice uniform, light corrosion on instruments.
Fig 2----Carbon steel files after being immersed in 5% NaOCl and collagen for ten minutes. Notice uniform, heavy corrosion.
Fig 3--Carbon steel files after being immersed in 5% NaOCl without collagen for ten minutes. Notice randomly distributed clumps of corrosion and large areas that appear to be unaffected.
of corrosion increased as the amount of collagen present increased, when the other factors remained constant. Because all root canals contain protein, this may be an important aspect to consider in clinical endodontics. Of course, a solution of purified bovine collagen is not the same solution of proteins that one would encounter in a root canal clinically. Nevertheless, the experimental solution was considered to parallel the clinical situation closely enough for the purposes of this study. The photographic technique used was sufficient to detect a real difference between stainless and carbon steel instruments. The ability of collagen to accelerate corrosion of stainless steel instruments could not be shown by photography. If the effect induced by collagen also applies to stainless steel, a more sensitive means of evaluation than photography would be required. The corrosion products have not been identified. They were submitted for analysis to obtain some sense of their protein content. 6 The Lowry and ninhydrin tests were used. Collectively, these tests sample for tryptophan, tyrosine, and alpha amino groups. The ninhydrin determination is capable of identifying appropriate substrates in quantities as small as 0.1/xmol. As such, these tests represent an extremely sensitive assay for protein. Yet when corrosion products were assayed, no protein was detected. Other known characteristics of these corrosion products are as follows: they are heavier than water; they appear to be insoluble in either water or NaOC1; and they are red brown in color. It is likely that they are oxides of iron, but further tests would be required for confirmation. Considering the friability of the corrosion products, one should be concerned with the deposition of these products in the canals and periapical
337
JOURNAL OF ENDODONTICS [ VOL 2, NO I1, NOVEMBER 1976
the carbon steel instruments were always greater in the groups ,that contained collagen than in those that did not. The greatest corrosion was seen when the combination of 5% NaOCI and collagen was used. Although purified bovine collagen is not the same type of protein encountered in root canals clinically, it was considered an adequate parallel for the purpose of this study.
Fig 4---Carbon steel files after ten minutes exposure to NaOCI without collagen: left file, 1% NaOCl; right file, 5 % NaOCl (orig mag • 10).
*Kerr Manufacturing Co., Romulus, Mich. tClorox Co., Oakland, Calif. ~Sigma Chemical Corp., St. Louis. The authors gratefully acknowledge the assistance of Drs. Charles Habib and Steven Davis for their advice during this study.
Fig 5 - - S a m e as in Figure 4 except collagen present in incubating solution with NaOCl: left file, 1% NaOCI plus collagen; right file, 5 % NaOCl plus collagen.
Drs. Eichner and Schoen are graduate students in endodontics; Dr. Goldman is clinical professor in endodontics and director of postgraduate endodontics, and Dr. Kronman is professor of orthodontics, Tufts University School of Dental Medicine, Boston. Requests for reprints should be directed to: Dr. Melvin Goldman, Tufts University School of Dental Medicine, 136 Harrison Ave, Boston, 02111. References
tissue, as well as their possible biologic consequences. The insolubility and specific gravity of these products would seem to make them difficult to remove. The role that corrosion products may play in endodontic failures is unknown. However, this variable can be minimized with the use of stainless steel instruments.
Summary Thirty-six stainless steel and 36 carbon steel endodontic instruments were immersed for ten minutes in solutions of 1% and 5 % NaOC1, with and without the addition of purified bovine collagen. Water, with and withotlt the
338
collagen, served as a control. The instruments were washed, dried, and then photographed at an image ratio of 1:1. The photographs showed no visible corrosion of the stainless steel instruments under any of the experimental conditions, nor did they show any visible corrosion of the carbon steel instruments when they were immersed in either water, water and collagen, or 1% NaOC1 without collagen. The photographs also showed corrosion on carbon steel instruments immersed in 1% NaOC1 and collagen, 5% NaOCI, and 5% NaOC1 and collagen. The amounts of corrosion on
1. Craig, R.G., and Peyton, F.A. Physical properties of carbon steel root canal files and reamers. Oral Surg 15:213 Feb 1962. 2. Craig, R.G., and Peyton, F.A. Physical properties of stainless steel endodontic files and reamers. Oral Surg 16: 206 Feb 1963. 3. Craig, R.G.; Mcllwain, E.D.; and Peyton, F.A. Bending and torsion properties of endodontic instruments. Oral Surg 25:239 Feb 1968. 4. Gutierrez, J.H.; Gigoux, C.; and Sanhueza, I. Physical and chemical deterioration of endodontic reamers during mechanical preparation. Oral Surg 28:394 Sept 1969. 5. Oliet, S., and Sorin, S.M. Cutting efficiency of endodontic reamers. Oral Surg 36:243 Aug 1973. 6. Blackburn, S. Amino acid determination; methods and techniques. New York, Marcel Dekker, Inc., 1968, p 69.