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Evaluation of enhanced vascular permeability of endodontic medicaments in vivo
JOURNAL OF ENDODONTICS I VOL 3, NO 9, SEPTEMBER 1977
E v a l u a t i o n of e n h a n c e d v a s c u l a r p e r m e a b i l i t y of e n d o d o n t i c medicaments
in v i v o
Michael Rutberq, DlVlD- Elisabeth S p a n q b e r g , DMD; a n d Larz Spancjberq. DDS, PhD, Farmington. C o - -
A technique for a s s a y i n q the a m o u n t of v a s c u l a r l e a k a q e incurred a s a result of intradermal injection of endodontic irriqants is described. The d a t a d e r i v e d from this methodoloqy, in addition to in vitro findinqs, should b e of great v a l u e in the selection process of efficient yet nonirritatinq e n d o d o n t i c medicaments.
Endodontic medicaments have been extensively evaluated in regard to their toxicity as determined by in vitro experimentationJ -s There have been no attempts to correlate in vitro findings with the actual effect of these agents on living tissues. Up to this point, the in vivo evaluation of these materials was basically studied via implantation and subsequent histologic examination of the affected tissues. 6"9 This certainly has led to considerable information about these materials, but there are certain limitations and problems concerned with such studies. It is difficult to quantitate the degree of in-
flammation; a medicament has its major effect on the tissues within hours or days. It is, therefore, difficult to sort out the inflammation that arises from the surgical trauma of implantation vs the inflammation from the material itself in a shortterm histologic study. It would seem, therefore, that a methodology that could quantitatively evaluate the tissue irritancy of endodontic medicaments would be of significant value. One of the cardinal signs of the acute inflammatory response is an increase in vascular permeability, which occurs when an injurious agent comes into contact with host tissue. This change occurs almost instantaneously after insult and may vary with the nature of the irritant. The vessels that are primarily involved are the postcapillary venules and capillaries. Leakage may occur due to direct damage of blood vessels and also due to release of chemical mediators such as histamine from host tissue. In healthy tissue, the blood tissue barrier is freely permeable to water, electrolytes, and small molecules but only slightly permeable to proteins.
The increase in vascular permeability in injured tissue is reflected by the passage of plasma proteins out of the vessels into the extravascular tissue. It has been generally accepted that some vital dyes such as Evans blue, trypan blue, or pontamine sky blue when given intravenously bind to plasma proteins, especially to albumin. Therefore, the accumulation of such dyes in sites that have incurred some type of injury indicates exudation of plasma proteins. The inflammatory potential of endodontic medicaments by such a technique has been reported. 10 However, at the time of that study there was no methodology available that could effectively quantitate the amount of leakage of dye. A subjective evaluation of the degree of leakage could only be made. Udaka, Takeuchi, and Movat 11 have reported a methodology that could quantitate such a response by extractirlg the dye and evaluating the amount of leakage via spectrophotometric analysis. The aims of the present study were to evaluate the usefulness of the method described by Udaka, Takeuchi, and Movat 11 in the study of chemical damage to tissue and, if
347
JOURNAL OF ENDODONTICS [ VOL 3, NO 9, SEPTEMBER 1977
necessary, modify the technique. In this evaluation process, some endodontic medicaments were used that normally are intended for root canal irrigation.
MATERIALS AND METHODS Animals White male Sprague-Dawley rats weighing approximately 200 g were anesthetized intraperitoneally with ketamine hydrochloride (10 mg/100 gm body weight). Their backs were shaved, and eight experimental sites were designated (Fig 1). Four additional areas of the back were used for positive and negative controls.
Test Materials
'q34qC2
Fig I--Experimental sites on dorsal sur/ace o] rat.
The test solutions used were all diluted in Ringer's solution. Stock solutions used were EDTAC* (according to Nygaard-~stby), 0.1% Zephiran,f 0.04% Iodopax,:~ 1% sodium hypochlorite, 5% sodium hypochlorite (bleach), and 3 % hydrogen peroxide. In addition, an experimental antimicrobial agent designated as "solution S" was tested. Procedures Intradermal injections of 0.1-ml test solution in various concentrations were given into the experimental sites (Fig 2). The negative control used was Ringer's solution and the positive control was 1% (w/v) phenol in Ringer's solution. One hour after the injection of the test solution, a 2% (w/v) solution of Evans blue dye in Ringer's solution was injected intravenously into the dorsal penile vein.
Two hours after the injection of the dye, the animals were killed by cervical dislocation. The dorsal skin was dissected away and tacked down on a cutting board. The skin lesions were punched out with a standard steel punch (14-ram diameter). Each piece of skin continuing the lesion was submerged in 4 ml of formamide (Baker)w and incubated at 45 C for 72 hours. After incubation, the solutions were filtered through a membrane filter (Whatman)[[ and the optical density of the filtrate was measured at 620/zm in a spectrophotometer. 82The total amount of dye was calculated by means of a standard calibration curve.
RESULTS The results are summarized in Table 1 and Figure 3. As would be
30-
uJ 20-
x 1~ NaOCI o 0.04~ Iodol~x 9 0.17.Zephiran z~ EDTA~
\ \ \
~
10-
Fig 2--Experimental site immediately a/ter six injections o] irritants.
O-
i
112
i
114
i
118
i
1116
t
I132
DILUTION OF STOCK SOLUTION Fig 3--Amount o/dye extracted .from injection sites three hours after application o] medicaments.
848
JOURNAL OF ENDODONTICS I VOL 3, NO 9, SEPTEMBER 1977
Fig 4---Top: outer surface o/experimental sites alter injection o/various irritants and intravenous injection o/blue dye. Bottom: same experimental sites in Figure 2 after dissection o/skin to expose entire lesions. A, B, C, and D re/er to corresponding lesions.
expected, increasing dilutions of test solutions gave decreasing values of dye leakage. However, the degree and rate of decrease with increasing dilution varied with the specific material. It was difficult to evaluate the amount of capillary leakage as judged from the external skin surface. T he size of the externat blue zone did not correspond to the size of the lesion in the underlying connective tissue when dissected free (Fig 4). The 5% solution of sodium hypochlorite and the hydrogen peroxide could not be evaluated due to the severe tissue damage incurred. The most revealing dilution of solutions appeared to be at one eighth, where we saw that the amount of leakage fell within the range of 4/zg to 13/~g. The most irritating materials were EDTAC, according to Nygaard0stby, and the quarternary ammonium compound, Zephiran. Slightly less irritation was caused by the 1% solution of sodium hypochlonte. Remarkably less irritation was produced by the iodophor, Iodopax. The decrease in irritating potential when solutions were diluted was less noticeable for the 1% solution of sodium hypochlorite as compared with the other irrigation medicaments (Table 1).
Table 1 " Medicaments used as irrigation solutions and amount of d y e in microqrams extracted from injection sites three hours after application of irrigant (X .4-SE).
Medicaments & Concentration of stock solution Dilution of stock solution 1/2 1/4 1/8 1/16 1/32 1/64
No.*
NaOCI 1%
0 . . . 20 35.6_+2.2 20 10.5_+1.1 20 8.9-+0.6 20 5.6-+0.2 20 3.2_+0.4
No.
Iodopax 0.04%
0 . . . 20 22.2_+2.0 20 6.1_+0.7 20 3.1-+0.3 20 1.5-+0.2 20 1.0_+0.2
No.
Zephiran 0.1%
0 . . . 0 . . . 20 13.0___1.0 20 3.5_+0.4 20 2.2--+0.3 0 . . .
No. EDTAC 0 . . . 0 . . . 20 13.1_+1.2 20 8.2_+0.4 20 1.6+_0.2 20 1.7_+_0.3
No.
Solution S
20 22.1_+2.4 20 13.5_+1.1 16 4.7_+0.3 16 3.1-+0.2 20 2.9_+0.2 20 2.9_+0.3
* Number of experiments.
349
JOURNAL O F E N D O D O N T I C S I VOL 3. N O 9, SEPTEMBER 1977
Table 2 9 Effect on vascular permeability after intradermal injections of 0.1 ml of R i n g e r ' s salt solution or phenol in salt solution a s m e a s u r e d in microqrams of dye. V a r i a b l e t i m e (t) b e t w e e n injection of irritant and dye. Experimental time w a s two hours -4- t (X +_SE).
Time (rain) Test solution Ringer's Phenol 0.50% Phenol 0.25%
No.*
0
8 5.0+0.4 8 11.5• 8 4.8•
No. 8 0 0
15 1.6• . . . . . .
No.
30
8 0.8+-0.3 8 8.7-+1.0 8 2.0+_0.6
No.
45
8 0 0
0.9• . . . . . .
No.
60
8 0.4• 8 9.9+_2.2 8 1.9•
No.
120
0 8 8
7.0+1.4 0.7•
. . .
* Number of experiments. Table 3 9 Effect on capillary permeability after intradermal injections of phenol in various dilutions. Experimental t i m e is three hours (X •
Dilution of stock solution
No.*
ptg of dye
1 1/2 1/4 1/8
20 40 40 20
62.7 • 5.0 26.2• 2.1• 0.9•
Fig 5--Connective tissue lesion after injection o] EDTAC, three hours a/ter experiment. Notice accumulation of extravasated red blood cells (arrows).
* Number of experimems.
I n comparison with the other materials, solution S had a remarkably low irritancy. DISCUSSION In the evaluation and modification of the methodology, it was found that a 60-minute interval between intradermal injection of the irritant and intravenous injection of dye was necessary. By this modification, the quantitation of vascular leakage caused by the irritant itself can be determined without being distorted by the trauma of the injection. As shown in Table 2, injection of Ringer's solution caused virtually no leakage after 60 minutes. On the contrary, injection of an irritating substance such as 0.5% phenol showed considerable leakage in the same time interval, which was solely due to chemical damage. It is possible within certain limits to obtain a dose-response curve with this technique (Table 3). There is an unreliable correlation between dose and response when the irritant induces insignificant capillary leakage. Thus,
850
it was found that when the amount of extracted dye was below 3/xg per lesion, the variation in response to an irritant was difficult to differentiate statistically from the normal tissue leakage occurring during routine experimentation. Unreliable correlation also was found when the irritant caused excessive capillary leakage resulting in accumulation of more than 20/xg of dye in the tissue sample. A good dose-response relationship was observed in the range of 3/xg to 20/zg of dye in the experimental site. The slope of the response curve varies with the individual material. A knowledge of the steepness of the slope may aid considerably in adjusting the proper dilution of a given material or in selecting between materials so as to give minimal tissue irritancy. A good example of this would be the comparison of decrease in tissue irritancy as seen when Zephiran and E D T A C are diluted (Table 1). A one-eighth
dilution of Zephiran 0.1% and E D T A C yields about the same amount of leakage of dye. However, when the dilution is increased to one sixteenth, E D T A C yields about 2.5 times the amount of dye. This methodology is not applicable if the irritant is too destructive, because the thin tissue around the test site easily deteriorates. In pilot studies using 5% sodium hypochloride (bleach), the tissue damage was so extensive that the affected tissue was necrotized and dissolved. In another experiment, a 3% hydrogen peroxide solution was evaluated. Extensive hemolysis and vasorhexis occurred, making it impossible to assay vascular leakage accurately. When studying higher concentrations of EDTAC, extravasated red blood cells are observed (Fig 5). This was an exclusive feature of EDTAC. It could possibly be due to the chelating effect on calcium ions as-
JOURNAL OF ENDODONTIC5 [ VOL 3, NO 9, SEPTEMBER 1977
sociated with the endothelial cells in the blood vessel walls. This vascular damage could result in increased accumulation of blue dye in the tissue not directly associated with enhanced permeability. In this case, however, the amount of extravasated dye due to capillary disruption could not have had a significant influence on the recordings, because the amount of r red blood cells are negligible when compared with the lesion (Fig 5). Thus, the results have to be carefully monitored in each experiment to ensure that enhanced permeability is measured. If these precautions are taken, the described modified technique could possibly have wider application in the evaluation of all dental materials used in opposition to connective tissue.
selection process of efficient and yet nonirritating endodontic medicaments. * N-O Therapeutics Hd, Oslo, Norway. "t Zephiran Chloride, Winthrop Labs., Div. of Sterling Drug, Inc., New York. $ Ferrosan, Malmo, Sweden. w J.T. Baker Chemical Co., Phillipsburg, NJ. II W. & R. Balston Ltd., England. 82Bausch & Lomb, lne., Rochester, NY. Dr. Rutberg is a former resident in endodontics at the University of Connecticut Health Center and is currently in private practice in Waterbury, Corm; Dr. E. Spangberg is assistant clinical professor, department of restorative dentistry; and Dr. L. Spangberg is professor of endodontics, School of Dental Medicine, University of Connecticut Health Center, Farmington. Requests for reprints should be directed to Dr. Larz Soangberg, Department of Endodontic~, School of Dental Medicine, University of Connecticut Health Cemer, Farmington, 06032.
SUMMARY
A technique assaying the amount of vascular leakage incurred as a result of intradermal injection of endodontic irrigants has been described. The data derived from this methodology, in addition to in vitro findings, should be of great value in the
References
1. Engstr/Sm, B., and Spangberg, L. Studies on root canal medicaments. 1. Cytotoxic effect of root canal antiseptics. Ac.ta Odontol Scand 25:77 June 1967. 2. Bengmark, S., and Rydberg, B. Cytotoxic action of cationic detergents on tissue growth in vitro. Acta Chir Seand 134:1, 1968.
3. Engstrfm, B., and Spangberg, L. V. Toxic and antimicrobial e{fects of antiseptics in vitro. Sven Tandlak Tidskr 62:543 Sept 1969. 4. Vander Wall, G.L., and Do~,vson, J. Antibacterial efficacy and cytotoxicity of three endodontic drugs. Oral Surg 33:230 Feb 1972. 5. Spangberg, L.; Engstr6m, B.; and Langeland, K. Biologic effects of dental materials. 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg 36:856 Dec 1973. 6. Coolidge, E.D. Reaction of dog tissue to drugs used in root canal treatment. JADA 19:747, 1932. 7. Torneck, C.D. Reaction of hamster tissue to drugs used in slerilization of the root canal. Oral Surg 14:730 June 1961. 8. Harrison, J.W., and Madonia, J.V. "Phe toxicity of parachlorophenol. Oral Surg 32:90 July 1971. 9. Poavell, D.L.; Marshall, F.J.; and Melfi, R.C. A histopa,thologic evaluation of tissue reactions to ehe minimum effective doses of some endodontic drugs. Oral Surg 36:261 Aug 1973. 10. Schilder, H., and Amsterdam, M. Inflammatory potential of roo~ canal medicaments. A preliminary report ineluding nonspecific drugs. Oral Surg 12:211 Feb 1959. 11. Udaka, K.; Takeuchi, Y.; and Movat, H.Z. Simple method for quantification of enhanced vascular permeability. Proc Soc Exp Biol Med 133:1384, 1970.
351
E v a l u a t i o n of e n h a n c e d v a s c u l a r p e r m e a b i l i t y of e n d o d o n t i c medicaments
in v i v o
Michael Rutberq, DlVlD- Elisabeth S p a n q b e r g , DMD; a n d Larz Spancjberq. DDS, PhD, Farmington. C o - -
A technique for a s s a y i n q the a m o u n t of v a s c u l a r l e a k a q e incurred a s a result of intradermal injection of endodontic irriqants is described. The d a t a d e r i v e d from this methodoloqy, in addition to in vitro findinqs, should b e of great v a l u e in the selection process of efficient yet nonirritatinq e n d o d o n t i c medicaments.
Endodontic medicaments have been extensively evaluated in regard to their toxicity as determined by in vitro experimentationJ -s There have been no attempts to correlate in vitro findings with the actual effect of these agents on living tissues. Up to this point, the in vivo evaluation of these materials was basically studied via implantation and subsequent histologic examination of the affected tissues. 6"9 This certainly has led to considerable information about these materials, but there are certain limitations and problems concerned with such studies. It is difficult to quantitate the degree of in-
flammation; a medicament has its major effect on the tissues within hours or days. It is, therefore, difficult to sort out the inflammation that arises from the surgical trauma of implantation vs the inflammation from the material itself in a shortterm histologic study. It would seem, therefore, that a methodology that could quantitatively evaluate the tissue irritancy of endodontic medicaments would be of significant value. One of the cardinal signs of the acute inflammatory response is an increase in vascular permeability, which occurs when an injurious agent comes into contact with host tissue. This change occurs almost instantaneously after insult and may vary with the nature of the irritant. The vessels that are primarily involved are the postcapillary venules and capillaries. Leakage may occur due to direct damage of blood vessels and also due to release of chemical mediators such as histamine from host tissue. In healthy tissue, the blood tissue barrier is freely permeable to water, electrolytes, and small molecules but only slightly permeable to proteins.
The increase in vascular permeability in injured tissue is reflected by the passage of plasma proteins out of the vessels into the extravascular tissue. It has been generally accepted that some vital dyes such as Evans blue, trypan blue, or pontamine sky blue when given intravenously bind to plasma proteins, especially to albumin. Therefore, the accumulation of such dyes in sites that have incurred some type of injury indicates exudation of plasma proteins. The inflammatory potential of endodontic medicaments by such a technique has been reported. 10 However, at the time of that study there was no methodology available that could effectively quantitate the amount of leakage of dye. A subjective evaluation of the degree of leakage could only be made. Udaka, Takeuchi, and Movat 11 have reported a methodology that could quantitate such a response by extractirlg the dye and evaluating the amount of leakage via spectrophotometric analysis. The aims of the present study were to evaluate the usefulness of the method described by Udaka, Takeuchi, and Movat 11 in the study of chemical damage to tissue and, if
347
JOURNAL OF ENDODONTICS [ VOL 3, NO 9, SEPTEMBER 1977
necessary, modify the technique. In this evaluation process, some endodontic medicaments were used that normally are intended for root canal irrigation.
MATERIALS AND METHODS Animals White male Sprague-Dawley rats weighing approximately 200 g were anesthetized intraperitoneally with ketamine hydrochloride (10 mg/100 gm body weight). Their backs were shaved, and eight experimental sites were designated (Fig 1). Four additional areas of the back were used for positive and negative controls.
Test Materials
'q34qC2
Fig I--Experimental sites on dorsal sur/ace o] rat.
The test solutions used were all diluted in Ringer's solution. Stock solutions used were EDTAC* (according to Nygaard-~stby), 0.1% Zephiran,f 0.04% Iodopax,:~ 1% sodium hypochlorite, 5% sodium hypochlorite (bleach), and 3 % hydrogen peroxide. In addition, an experimental antimicrobial agent designated as "solution S" was tested. Procedures Intradermal injections of 0.1-ml test solution in various concentrations were given into the experimental sites (Fig 2). The negative control used was Ringer's solution and the positive control was 1% (w/v) phenol in Ringer's solution. One hour after the injection of the test solution, a 2% (w/v) solution of Evans blue dye in Ringer's solution was injected intravenously into the dorsal penile vein.
Two hours after the injection of the dye, the animals were killed by cervical dislocation. The dorsal skin was dissected away and tacked down on a cutting board. The skin lesions were punched out with a standard steel punch (14-ram diameter). Each piece of skin continuing the lesion was submerged in 4 ml of formamide (Baker)w and incubated at 45 C for 72 hours. After incubation, the solutions were filtered through a membrane filter (Whatman)[[ and the optical density of the filtrate was measured at 620/zm in a spectrophotometer. 82The total amount of dye was calculated by means of a standard calibration curve.
RESULTS The results are summarized in Table 1 and Figure 3. As would be
30-
uJ 20-
x 1~ NaOCI o 0.04~ Iodol~x 9 0.17.Zephiran z~ EDTA~
\ \ \
~
10-
Fig 2--Experimental site immediately a/ter six injections o] irritants.
O-
i
112
i
114
i
118
i
1116
t
I132
DILUTION OF STOCK SOLUTION Fig 3--Amount o/dye extracted .from injection sites three hours after application o] medicaments.
848
JOURNAL OF ENDODONTICS I VOL 3, NO 9, SEPTEMBER 1977
Fig 4---Top: outer surface o/experimental sites alter injection o/various irritants and intravenous injection o/blue dye. Bottom: same experimental sites in Figure 2 after dissection o/skin to expose entire lesions. A, B, C, and D re/er to corresponding lesions.
expected, increasing dilutions of test solutions gave decreasing values of dye leakage. However, the degree and rate of decrease with increasing dilution varied with the specific material. It was difficult to evaluate the amount of capillary leakage as judged from the external skin surface. T he size of the externat blue zone did not correspond to the size of the lesion in the underlying connective tissue when dissected free (Fig 4). The 5% solution of sodium hypochlorite and the hydrogen peroxide could not be evaluated due to the severe tissue damage incurred. The most revealing dilution of solutions appeared to be at one eighth, where we saw that the amount of leakage fell within the range of 4/zg to 13/~g. The most irritating materials were EDTAC, according to Nygaard0stby, and the quarternary ammonium compound, Zephiran. Slightly less irritation was caused by the 1% solution of sodium hypochlonte. Remarkably less irritation was produced by the iodophor, Iodopax. The decrease in irritating potential when solutions were diluted was less noticeable for the 1% solution of sodium hypochlorite as compared with the other irrigation medicaments (Table 1).
Table 1 " Medicaments used as irrigation solutions and amount of d y e in microqrams extracted from injection sites three hours after application of irrigant (X .4-SE).
Medicaments & Concentration of stock solution Dilution of stock solution 1/2 1/4 1/8 1/16 1/32 1/64
No.*
NaOCI 1%
0 . . . 20 35.6_+2.2 20 10.5_+1.1 20 8.9-+0.6 20 5.6-+0.2 20 3.2_+0.4
No.
Iodopax 0.04%
0 . . . 20 22.2_+2.0 20 6.1_+0.7 20 3.1-+0.3 20 1.5-+0.2 20 1.0_+0.2
No.
Zephiran 0.1%
0 . . . 0 . . . 20 13.0___1.0 20 3.5_+0.4 20 2.2--+0.3 0 . . .
No. EDTAC 0 . . . 0 . . . 20 13.1_+1.2 20 8.2_+0.4 20 1.6+_0.2 20 1.7_+_0.3
No.
Solution S
20 22.1_+2.4 20 13.5_+1.1 16 4.7_+0.3 16 3.1-+0.2 20 2.9_+0.2 20 2.9_+0.3
* Number of experiments.
349
JOURNAL O F E N D O D O N T I C S I VOL 3. N O 9, SEPTEMBER 1977
Table 2 9 Effect on vascular permeability after intradermal injections of 0.1 ml of R i n g e r ' s salt solution or phenol in salt solution a s m e a s u r e d in microqrams of dye. V a r i a b l e t i m e (t) b e t w e e n injection of irritant and dye. Experimental time w a s two hours -4- t (X +_SE).
Time (rain) Test solution Ringer's Phenol 0.50% Phenol 0.25%
No.*
0
8 5.0+0.4 8 11.5• 8 4.8•
No. 8 0 0
15 1.6• . . . . . .
No.
30
8 0.8+-0.3 8 8.7-+1.0 8 2.0+_0.6
No.
45
8 0 0
0.9• . . . . . .
No.
60
8 0.4• 8 9.9+_2.2 8 1.9•
No.
120
0 8 8
7.0+1.4 0.7•
. . .
* Number of experiments. Table 3 9 Effect on capillary permeability after intradermal injections of phenol in various dilutions. Experimental t i m e is three hours (X •
Dilution of stock solution
No.*
ptg of dye
1 1/2 1/4 1/8
20 40 40 20
62.7 • 5.0 26.2• 2.1• 0.9•
Fig 5--Connective tissue lesion after injection o] EDTAC, three hours a/ter experiment. Notice accumulation of extravasated red blood cells (arrows).
* Number of experimems.
I n comparison with the other materials, solution S had a remarkably low irritancy. DISCUSSION In the evaluation and modification of the methodology, it was found that a 60-minute interval between intradermal injection of the irritant and intravenous injection of dye was necessary. By this modification, the quantitation of vascular leakage caused by the irritant itself can be determined without being distorted by the trauma of the injection. As shown in Table 2, injection of Ringer's solution caused virtually no leakage after 60 minutes. On the contrary, injection of an irritating substance such as 0.5% phenol showed considerable leakage in the same time interval, which was solely due to chemical damage. It is possible within certain limits to obtain a dose-response curve with this technique (Table 3). There is an unreliable correlation between dose and response when the irritant induces insignificant capillary leakage. Thus,
850
it was found that when the amount of extracted dye was below 3/xg per lesion, the variation in response to an irritant was difficult to differentiate statistically from the normal tissue leakage occurring during routine experimentation. Unreliable correlation also was found when the irritant caused excessive capillary leakage resulting in accumulation of more than 20/xg of dye in the tissue sample. A good dose-response relationship was observed in the range of 3/xg to 20/zg of dye in the experimental site. The slope of the response curve varies with the individual material. A knowledge of the steepness of the slope may aid considerably in adjusting the proper dilution of a given material or in selecting between materials so as to give minimal tissue irritancy. A good example of this would be the comparison of decrease in tissue irritancy as seen when Zephiran and E D T A C are diluted (Table 1). A one-eighth
dilution of Zephiran 0.1% and E D T A C yields about the same amount of leakage of dye. However, when the dilution is increased to one sixteenth, E D T A C yields about 2.5 times the amount of dye. This methodology is not applicable if the irritant is too destructive, because the thin tissue around the test site easily deteriorates. In pilot studies using 5% sodium hypochloride (bleach), the tissue damage was so extensive that the affected tissue was necrotized and dissolved. In another experiment, a 3% hydrogen peroxide solution was evaluated. Extensive hemolysis and vasorhexis occurred, making it impossible to assay vascular leakage accurately. When studying higher concentrations of EDTAC, extravasated red blood cells are observed (Fig 5). This was an exclusive feature of EDTAC. It could possibly be due to the chelating effect on calcium ions as-
JOURNAL OF ENDODONTIC5 [ VOL 3, NO 9, SEPTEMBER 1977
sociated with the endothelial cells in the blood vessel walls. This vascular damage could result in increased accumulation of blue dye in the tissue not directly associated with enhanced permeability. In this case, however, the amount of extravasated dye due to capillary disruption could not have had a significant influence on the recordings, because the amount of r red blood cells are negligible when compared with the lesion (Fig 5). Thus, the results have to be carefully monitored in each experiment to ensure that enhanced permeability is measured. If these precautions are taken, the described modified technique could possibly have wider application in the evaluation of all dental materials used in opposition to connective tissue.
selection process of efficient and yet nonirritating endodontic medicaments. * N-O Therapeutics Hd, Oslo, Norway. "t Zephiran Chloride, Winthrop Labs., Div. of Sterling Drug, Inc., New York. $ Ferrosan, Malmo, Sweden. w J.T. Baker Chemical Co., Phillipsburg, NJ. II W. & R. Balston Ltd., England. 82Bausch & Lomb, lne., Rochester, NY. Dr. Rutberg is a former resident in endodontics at the University of Connecticut Health Center and is currently in private practice in Waterbury, Corm; Dr. E. Spangberg is assistant clinical professor, department of restorative dentistry; and Dr. L. Spangberg is professor of endodontics, School of Dental Medicine, University of Connecticut Health Center, Farmington. Requests for reprints should be directed to Dr. Larz Soangberg, Department of Endodontic~, School of Dental Medicine, University of Connecticut Health Cemer, Farmington, 06032.
SUMMARY
A technique assaying the amount of vascular leakage incurred as a result of intradermal injection of endodontic irrigants has been described. The data derived from this methodology, in addition to in vitro findings, should be of great value in the
References
1. Engstr/Sm, B., and Spangberg, L. Studies on root canal medicaments. 1. Cytotoxic effect of root canal antiseptics. Ac.ta Odontol Scand 25:77 June 1967. 2. Bengmark, S., and Rydberg, B. Cytotoxic action of cationic detergents on tissue growth in vitro. Acta Chir Seand 134:1, 1968.
3. Engstrfm, B., and Spangberg, L. V. Toxic and antimicrobial e{fects of antiseptics in vitro. Sven Tandlak Tidskr 62:543 Sept 1969. 4. Vander Wall, G.L., and Do~,vson, J. Antibacterial efficacy and cytotoxicity of three endodontic drugs. Oral Surg 33:230 Feb 1972. 5. Spangberg, L.; Engstr6m, B.; and Langeland, K. Biologic effects of dental materials. 3. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg 36:856 Dec 1973. 6. Coolidge, E.D. Reaction of dog tissue to drugs used in root canal treatment. JADA 19:747, 1932. 7. Torneck, C.D. Reaction of hamster tissue to drugs used in slerilization of the root canal. Oral Surg 14:730 June 1961. 8. Harrison, J.W., and Madonia, J.V. "Phe toxicity of parachlorophenol. Oral Surg 32:90 July 1971. 9. Poavell, D.L.; Marshall, F.J.; and Melfi, R.C. A histopa,thologic evaluation of tissue reactions to ehe minimum effective doses of some endodontic drugs. Oral Surg 36:261 Aug 1973. 10. Schilder, H., and Amsterdam, M. Inflammatory potential of roo~ canal medicaments. A preliminary report ineluding nonspecific drugs. Oral Surg 12:211 Feb 1959. 11. Udaka, K.; Takeuchi, Y.; and Movat, H.Z. Simple method for quantification of enhanced vascular permeability. Proc Soc Exp Biol Med 133:1384, 1970.
351