- Email: [email protected]
Citric and lactic acids as root canal irrigants in vitro
JOURNALOF ENDODONTICS ] VOL 5, NO 9, SEPTEMBER1979
Citric and lactic acids as root canal irrigants in vitro Blake E. W a y m a n , DDS, MS; William M. Kopp, DDS, MS; Gerald J. Pinero, PhD; and E. P. Lazzari, PhD, H o u s t o n
T h e efficacy of solutions of lactic acid, t h r e e c o n c e n t r a t i o n s of citric acid, s o d i u m h y p o c h l o r i t e , a n d p h y s i o l o g i c saline u s e d as root canal irrigants w a s e v a l u a t e d . W i t h u s e of extracted, s i n g l e - r o o t e d h u m a n teeth, the a m o u n t s of h y d r o x y p r o l i n e a n d c a l c i u m r e m o v e d b y these irrigants w e r e m e a s u r e d . T h e t r e a t e d teeth w e r e t h e n e x a m i n e d w i t h a s c a n n i n g e l e c t r o n m i c r o s c o p e . It w a s c o n c l u d e d t h a t a 10% s o l u t i o n of citric acid as a lubricant, f o l l o w e d b y a 2.5% s o l u t i o n of s o d i u m h y p o c h l o r i t e as a n irrigant, a n d t h e n a g a i n u s e of the citric acid solution, will p r o d u c e clean canal walls with p a t e n t d e n t i n a l tubules.
A 50% solution of sulfuric acid' was one of the first of the many agents that have been utilized as root canal irrigants. Proteolytic enzymes ~-4 and chlorine-releasing agenW '''~ have also been used. Sodium hypochlorite has been the most commonly used root canal irrigant since its introduction and is an effective solvent of organic tissue5 It is also an irritant to healthy tissue"-" and an effective germicidal agent. TI' Other irrigants such as potentiated 1,5 pentanedia[, '~ urea peroxide, ':~ Gly-oxide," and hydrogen peroxide have been used. Ethylenediaminotetraacetic acid (EDTA) was the first reported chelating agent to be used as a root canal irrigant. '~-''; Concentrations of E D T A as small as 15% dissolve hydroxyapatite at least as well as a 20% solution of hydrochloric acid or a 50% solution of sulfuric acid.'" Several studies using a scanning electron microscope 1:'-'~'~ have shown that chelating agents consistently produced patent dentinal tubules
258
and, often, the cleanest canal walls. Another chelating agent, citric acid, has been recently reported by Loci'" to be an effective root canal irrigant when used alternately with sodium hypochlorite. Because such chelating agents as lactic and citric acids occur naturally in the body, these solutions are thought to be more acceptable biologically than many irrigants that are commonly in use. The purpose of this study was to compare the effectiveness of solutions of lactic acid, various concentrations of citric acid, sodium hypochlorite, and physiologic saline (control) as organic and inorganic tissue solvents. Biochemical a,Csays were done and scanning electron micrographs were taken to assess the effects of the various irrigants on the canal walls.
M E T H O D S A N D MATERIALS Hydroxyproline assay The hydroxyproline assay in vitro used 120 permanent human single-
rooted teeth. Previous histories for these teeth were not available, but, generally, they were removed for orthodontic or periodontal reasons. The anatomical crowns were removed with an Isomet* low-speed saw, and each tooth was examined for the presence of an intact pulp. If no pulp tissue was observed, the tooth was discarded. Each canal was instrumented through the apex to a size 80 or 90 file, rinsed with 2 ml of de-ionized water, and stored in 100% humidity at 4 C until used. The root apex was sealed with wax, mounted on wax squares, and placed randomly in one of lhe six sample groups for irrigation. The six irrigant solutions used were 0.86% physiologic saline (control); 5.25% sodium hypochlorite (pH adjusted to 11.7); 50% citric acid, (w/v, p H 1.6, prepared in the laboratory); Epoxylite 9060t cavity cleaner (commercial solution of 50% citric acid); 50% lactic acid, (v/v, p H 1.5, prepared in the laboratory); and
JOURNAL OF ENDODONTICS I VOL 5, NO 9, SEPTEMBER 1979
5.25% sodium hypochlorite and laboratory-prepared 50% citric acid solution alternated in varying sequences. Each irrigant was tested on 20 teeth. After instrumentation, the irrigating solution was deposited in the canal to the level of the surface of the sectioned tooth, and was allowed to remain for five minutes. When solutions were alternated, each solution remained in the canal for 21/2 minutes. The solutions were removed from the canal using enough paper points to completely dry the canal surface. The paper points were washed three times with 2 ml of deionized water per wash, which was collected, slant frozen, and lyophilized. Then, 2 ml of 6N hydrochloric acid was added to each dry sample; these were capped and heated for three hours at 130 C. The hydrochloric acid hydrolysate was removed by flash evaporation; 3 ml of de-ionized water was added and removed in 1-ml increments to dissolve the dry sample. The samples were again slant frozen, lyophilized, and prepared for assay by adding 2 ml of de-ionized water. This was divided into two 1-ml samples, to which 1 ml of de-ionized water was added. A reagent blank and a hydroxyproline standard were determined for each group. The assay for hydroxyproline was done according to the method by Woessner,-'" whereas the method outlined by Stewart'-" was used to determine the volume of the canal. Calcium assay
Six groups of five single-rooted human teeth were prepared for the calcium assay in the same manner described for the hydroxyproline assay. The teeth of each group were filled with one of the following irri-
gant solutions, which were left in the canals for five minutes: 0.86% physiologic saline (control); 50% lactic acid (v/v, pH 1.5); 50% citric acid (w/v, pH 1.6); 25% citric acid (w/v, pH 1.6); 10% citric acid (w/v, pH 1.6); and 5.25% sodium hypochlorite (Clorox) with the pH adjusted to 11.7. The samples for the calcium assay were collected and washed in the same manner described for the hydroxyproline assay. The l-ml samples from groups 1 and 6 were diluted with 1 ml of a lanthanum oxide solution (1,200 ppm). Groups 2, 3, 4, and 5 were diluted with 1.6 ml of the lanthanum oxide solution for each 0.4 ml of sample, because of higher concentrations of calcium in these groups. The optical density for each sample of known and unknown concentration was determined with an atomic absorption apparatus.~ 2~ Electron microscope
Canals of teeth selected for examination with the scanning electron microscope were prepared in the manner previously described for the biochemical assays. Three teeth from each of the following seven groups of solutions were examined: 0.86% physiologic saline; 50% lactic acid; 50% citric acid; 25% citric acid; 10% citric acid; 5.25% sodium hypochlo-
rite; and 50% citric acid used as a lubricant during instrumentation. The irrigants remained in the canals for five minutes, and were removed using paper points. The canals were then rinsed with 10 ml of de-ionized water delivered by needle and syringe, and were sealed with boxing wax. The teeth were notched buccally and lingually with a no. 700 tapered fissure bur in a high-speed handpiece and then were split longitudinally. The samples were dried in increasing concentrations of ethanol and then in amyl acetate. Carbon dioxide served as the transitional solvent in the critical point-drying apparatus.w The specimens were mounted on studs with a conductive adhesive and then were coated with a 500 A layer of gold in a sputter coater.II Photomicrographs of typical areas were taken at magnifications of 60 to 1,500 diameters. 82 The canals were evaluated for the appearance of debris, patent dentinal tubules, and residual pulp tissue. RESULTS The control group in the assay for hydroxyproline was tested with a physiologic saline solution irrigant that yielded no measural~le quantity of hydroxyproline (Table 1). The control irrigant yielded an average of
Table I. Comparison of all groups for hydroxyproline.
Sample group
Z,#g
~ #g
2 vol
Saline 5.2% hypochlorite 50% citric acid 50% lactic acid Epoxylite Combination-Varied Sequence hypochlorite/citric acid citric acid/hypochlorite
0.00 112.34 16.29 10.12 13.68 85.68
0.00 2.80 0.40 0.25 0.34 2.14
20.74 21.03 19.15 14.38 20.92 18.19
~/~g//zl 0.000 0.410 0.021 0.020 0.016 0.122
SD/~g/p.l 0.000 0.076 0.025 0.021 0.021 0.062
40.70 44.98
2.04 2.24
18.32 18.07
0.127 0.117
0.068 0.056 259
J O U R N A L OF E N D O D O N T I C S
Table 2 9 Comparison
Sample group 1.11 /~g//~l of calcium as determined from a 1-ml sample (Table 2). Scanning electron micrographs of the canals treated with saline solution disclosed a smeared layer probably consisting of organic and inorganic debris with no readily visible dentinal tubules (Fig 1). The sample group irrigated with the 5.25% solution of sodium hypochlorite yielded the greatest amount of hydroxyproline (0.14/~g/ ~1), which was significantly greater (P < .001) than all groups except the combination group, which yielded 0.12 /zg//fl (Table 1), The calcium assay for sodium hypochlorite gave a mean of 1.08 /~g/~l (Table 2). The scanning electron micrographs showed canal walls with a smeared layer of material (Fig 2) similar in appearance to that produced by physiologic saline solution. Some dentinal tubules were visible, but, in general, the instrumented canal surface was rough and irregular. In an uninstrumented area (Fig 3) globular dentin with patent dentinal tubules was seen, with apparently no organic matter present. The hydroxyproline assay for the group treated with 50% lactic acid solution produced results that were significantly different from the control (P < .001) with a mean of 0.020 ftg/~l (Table 1). In the calcium assay, lactic acid removed a mean amount of 8,82 ftg/~tl, which is also highly significant ( P < .001) when compared with the saline control. Scanning electron micrographs showed clean canal walls, but the dentinal tubules did not appear completely patent (Fig 4). The 50% citric acid solution prepared in the laboratory and the preparation of epoxylite 9060 yielded 0.021 and 0.016 /~g/btl of hydroxy260
Saline 50% lactic acid 50% citric acid 25% citric acid 10% citric acid Sodium hypochlorite ~Five
teeth per
group
I
V O L 5, N O 9, SEPTEMBER 1979
o f all g r o u p s f o r c a l c i u m . *
Y.~g
X/a,g
~ vol
,~ ~g//,tl
S.D. /.tg//d
101.96 749.02 970.50 708.88 697.20 90.03
20.39 149.80 194.10 141.78 139.44 I0.01
17.92 17.33 21.79 14.57 20.15 17.22
1.11 8.82 8.92 10.17 7.82 1.08
0.53 0.56 0.68 1,63 2.92 0.34
per five minute.
.
i,i
...
~
".
(~
F(r l-Instrumented root canal irrigated with physiologzc saline; canal appears oh'an, but ,o patent dentinal tubules are uisible. Some gross debris ('D ) is present.from splitting lechnique (o~g mag X 1,250).
t d, t
i
.
r
. '
9
.;r
5
.
9
,i
"~ ..
J
: *..
.
'
i
. ~,
t.. .
Fzig 2-Canal treated with sodium /?rpochlorile. Instrumented area shows no organtc debris. Rough. smeared appearance is thoueht to be tnot~amc material resulting from inslrU mentation, l)entinal tubules are z'isible, Ira/ occluded (orig mag x 1,500). ,, ~
.t~
. . ~.,r
. ,..
:...t, .
JOURNAL OF ENDODONTICS I VOL 5, NO 9, SEPTEMBER 1979
Fzg 5--Canal treated with 10% solution of citric acid. A rea shown is about mid-root and has eroded appearance seeming to e.~pose dentinal matrix.fiber (arrow). Dentinal tubules are patent and canal waU appears clean. Some debris (D) is present (ortg mag • 1,500).
'
Fig 3--Uninstrumented area of canal treated with sodium hypochlorite in which dentinal tubules are visible and no organic tissue can be distinguished. Irregular surface (G) is thought to be inorganic globular dentin (orig rnag X 1,500).
c
(
~
"
i~ ' ~
,,
{ t'
FtIr 4-Canal treated with lactic acid appears clean with obvious dentinal tubules, some of which are open. whereas others appear occluded (orzir mag • 1,250).
.h
~
.
l~'ti~ 6--Canal treated with 5(P/~ solution of citric acid. Dentinal tubules (7") are wide open and tooth su!filce is smooth and clean. Area has been instrumented (orzg mag • 1,500).
).
4..,
k ."N .
.
t"i ,
; a..,
proline (Table 1), respectively, with no statistically significant difference. This is, however, significantly more (P < .001) than the control group. T h e calcium assay showed a highly significant difference (P < .001) for all three concentrations of citric acid when compared with the control. T h e mean for the group treated with a solution of 50% citric acid was 8.92/~g//~l; 10.17/ug//~l for the group treated with a 25% solution of citric acid, and 7.82 /ag//~l for the group that received a 10% solution of citric acid, (Table 2). Micrographs of the teeth irrigated with the three concen-
trations of citric acid showed canal walls that were generally free of the smeared appearance. There were more patent tubules with even a 10% concentration of citric acid (Fig 5) than with the sodium hypochlorite. The teeth treated with 50% citric acid (Fig 6) had dentinal tubules that were generally more patent, and canal walls that were cleaner, than those treated with the 10% and 25% concentrations. However, there was a great variation in the patency of the tubules at the lower concentrations. Figure 7, taken near the cementoenamel junction, shows that citric 261
JOURNAL OF ENDODONTICS I VOL 5, NO 9, SEPTEMBER 1979
acid had little effect on the organic matrix. The alternation of sodium hypochlorite and 50% citric acid solution yielded 0.12 ffg/ffl of hydroxyproline, which was significantly more (P < .100) than the amount yielded by the control (saline solution) or chelating agents, but less than that yielded by the group treated with 5.25% sodium hypochlorite solution. The sequence of application of the solution was varied, but there was no significant difference in the results of the assay relating to the sequence in which the agents were used. Scanning electron micrographs of the teeth in which the 50% solution of citric acid was used as both a lubricant and irrigant showed a relatively smooth, clean canal surface with patent dentinal tubules in the instrumented areas (Fig 8). In the uninstrumented areas, the dentinal tubules were patent, and cells, which may be odontoblasts and fibroblasts, were present (Fig 9).
Fzg 7-Canal treated with 50% solution of citric acid, near the cementoenamel junction in instrumented area. Undestrored fibers (F) remain. This is thought to be area in which calcification was possibly occurring. Note how fine matrix fibers have been exposed (arrow) (orzg mag X 1,500).
#J P
"4"'
..
DISCUSSION Analysis of amino acids in dentin protein has shown that much more hydroxyproline is present in dentinal collagen than in other types of collagen. '-':~ Because collagen is a major component of dentin, the measurement of hydroxyproline removed from dentin is an ideal method of determining the effect of the irrigants on the dissolution of organic material of the dentin and pulp. The assay for hydroxyproline according to Woessner'-'" has been shown to be accurate and simple for small amounts of this amino acid. The canal volume of each of the teeth was different and would have given greater amounts of hydroxyproline for a larger canal. For this reason, the 262
,,a;. ~J .F~
.
~.~. t
,, ..j
"
.q r
.
Fig 9-Root canal in which 50% solution of citric acid'was used as lubricant. Uninstrumented area near CI:.J shows cellular fibrous components (C). Note clean, open dentinal tubules (7~) (orig mag x 800).
,,..r.
9
}
/'
b'zg 8--Root canal in which 50% solution of citric acid was used as lubricant. Canal su~lce appears clean with hzgh(r ~,isible patent dentinal tubules. Instrument marks (1) are present," no smeared layer is seen (ortlg mag • 1,400).
JOURNAL OF E N D O D O N T 1 C S
VOL 5, N O 9, SEPTEMBER 1979
COMPARISON OF ALL GROUPS FOR HYDROXYPROLINE ~- ug/ul 0.14 0.13 0.12
/ ,/jZp ///
0.11
H;':"; .'//.." /.../
0.10
.
'5,
.
'
..~
0.09 ,'//.
0.08
,
--,;.,>..
.
.
0.07
/'/:, I/:,
",,
0.06
>;/',,-:: ,/:
'
:q,;).,)
0.05 ' 0.04 '
"V,>#.;..
0.03 '
/,,>~
0.02
,////
."
.
.;;...;.
. ;
Fq~ lO-,'ffean calm's (y~,lff/) ?lhldro.wpr,dim' rcmoccd br z,ar/ou.s Jrtlgant.~ during.licr-mmuh' peri~M
,, : .;:"; i :
..;,.;..:
<'9 9:9149149149
1"' '".';i;;
0.01 ( ,/i.
PHYSIOLOGIC
50%
50%
EPOXYLITE
SALINE
LACTIC ACiD
CITRIC ACID
9060
/r
SODIUM
COMBINATION-
HYPDCHLORITE VARIED SEQUENCE
COMPARISON OF ALL GROUPS FOR CALCIUM ug/ul 11 10
",..;;
9 '
!'/ / /
,.J
/;
/
/
I
/ ' , "1
8 7
"/
6
k'(Z" 11 Mean r (ffgltd) qf calcium remot,ed by cartous irr(gants during rice-minute per,od.
9
J
,'/
;"/..y'/2~
//./...//.
5 4
/. . . . . . . /./'...
-,.///
3
///
2
.,/...
""'~--//;"""1
-q
)..-,
' : Z ' i . / .. .
.,.-
I
volumes of the canals were determ i n e d a c c o r d i n g to the m e t h o d of Stewart. :~ In the h y d r o x y p r o l i n e study, the physiologic saline did not remove a m e a s u r a b l e a m o u n t , a n d it was shown t h a t the effect o f the 50% solutions of citric a n d lactic acid on the dissolution of organic tissue is also m i n i m a l . However, the h y d r o x y proline dissolved by the sodium hypochlorite was seven times more than the a m o u n t r e m o v e d by the chelating agents (Fig 10). In the calcium analysis, lactic acid and the three concentrations of citric acid removed seven to nine times as
;1
9 ;. • <; %.
;, ,,. . .:,.. .:,..,. .....
PHYSIOLOGIC
50%
50%
26%
10%
SALINE
LACTIC ACID
CITRIC ACID
CITRIC ACID
CITRIC ACID
much c a l c i u m as the physiologic saline control and the sodium h y p o c h l o r i t e (Fig 11). T h e 25% solution of citric acid removed slightly more c a l c i u m than the other chelating solutions tested. However, this value was not statistically significant (P < .05), a n d the a p p a r e n t increase is t h o u g h t to be due to the small n u m b e r of teeth tested in each group. T h e statistical analysis indicates a considerable deviation from the mean in m a n y of the groups, p e r h a p s because of the wide range of patients from w h o m these teeth were obtained. T h e results of the h y d r o x y p r o l i n e
G:;:~
SODIUM
HYPOCHLORITE
a n d calcium assays indicate that sodium h y p o c h l o r i t c is a much better solvent of organic tissue c o m p o n e n t s than are the c h e l a t i n g agents, citric acid a n d lactic acid. IInwcver, sodiu m hypochlorite has litth" effect on the inorganic m a t e r i a l tbund within the dentin 9 Scanning electron m i c r o g r a p h s showed that all concentrations of citric and lactic acid p r o d u c e d much cleaner canal walls and more p a t e n t d e n t i n a l tubules than the saline control or sodium hypochlorite. T h e d e n t i n a l particles seen in all the groups e x a m i n e d are thought to result from the techniques used in 263
JOURNAL OF E N D O D O N T 1C S I VOL 5, N O 9, SEPTEMBER 1979
micrographs studies are consistent with the results of the biochemical tests done in this study. In contrast, the micrographs of the teeth treated with sodium hypochlorite (Fig 2, 3) show no fibrous or cellular tissue and indicate that the solvent action of this irrigant is on the organic tissue of the root canal. The ideal irrigant is thought to be one that is germicidal, dissolves organic and inorganic tissue, and can be quickly metabolized with no adverse effect should it be accidently expressed beyond the apex. The germicidal effect of sodium hypochlorite has been well established, and its ability to dissolve organic tissue and cellular debris in the root canal has also been demonstrated7 The irritation to normal tissues by sodium hypochlorite, however, remains a concern. ~-'' Citric and lactic acid occur naturally in the body. Citric acid is found in mitochondria '-':' and is used by blood banks to prevent coagulation.'-'" Citric acid has also been shown to be the most effective acid in altering the solubility of hydroxyapatite. '-'~ Lactic acid occurs in the muscles under anaerobic conditions during vigorous muscular exerciseP :' Dentinal permeability to microorganisms is also of concern to the endodontist; 1''-'~ open dentinal tubules are thought to allow intracanal medicaments to enter and destroy any bacteria that may be present, '-'~:~~ Some investigators have found that chelating agents increase the dentinal permeability,'" whereas others reported a reduction.:" The results of this and another study :~: suggest that the use of two irrigants would yield the best results. Initially, a 10% solution of citric acid as a lubricant and hydroxyapatite solvent should be used, which helps 264
to maintain patent dentinal tubules. This should be followed by a 2.5% solution of sodium hypochlorite applied for five minutes :l'-'with frequent changes to dissolve the remaining organic material and destroy any microorganisms that may be present. Further instrumentation with a 10% solution of citric acid is recommended to ensure that the canal walls are clean and that the dentinal tubules are patent. It is believed that the use of a 10% solution of citric acid and a 2.5% solution of sodium hypochlorite, as described, removes almost all organic and inorganic debris from the canal. The scanning electron micrographs indicate that relatively germfree and clean canals with patent dentinal tubules are produced. This may be important clinically in allowing intracanal medicaments to be more effective and in permitting better obturation with the filling materials available. SUMMARY Concentrations of 10%, 25%, and 50% citric acid and 50% lactic acid were used as root canal irrigants to determine their effectiveness as tissue solvents in vitro. These solutions were compared with solutions of sodium hypochlorite and a saline control. The hydroxyproline values were used to indicate the solvent action of the irrigating solutions on the organic components of the tooth. Results show that the 5.25% sodium hypochlorite solution produced seven times more hydroxyproline than the preparations of citric acid or lactic acid, indicating that sodium hypochlorite produces a significantly greater ( P < .001) dissolution of organic tissue. The calcium values determined for all test agents indi-
cated the effect of these solutions on the inorganic dentinal components. Results showed that the solutions of citric and lactic acid all dissolved seven to nine times as much calcium as the sodium hypochiorite and the saline control, and this difference was f o u n d to be significant (P < .001). The solutions of citric and lactic acid were thought to act as chelating agents. The effects of the various irrigants on surfaces of the root canals were evaluated with the scanning electron microscope. All solutions of citric and lactic acid used only as an irrigant after dry instrumentation produced cleaner, smoother walls than did the solutions of sodium hypochlorite or the saline control. The 10% concentration of citric acid opened up the dentinal tubules and cleaned the canal wall, but not as effectively as the stronger concentrations of citric acid, although it was equal in the ability to dissolve minerals. Lactic acid left a film on the surface, possibly because of its greater viscosity and incomplete removal. Combined with instrumentation, the citric acid produced clean canal walls and patent dentinal tubules. The sodium hypochlorite produced a cleaner canal surface than did the saline control, but did not remove the debris from the dentinal tubules that resulted from the instrumentation of the root canal. The patent tubules and clean canal walls may be desirable clinically in allowing greater penetration of intracanal medicaments or filling materials into the dentinal tubules.
*Buehler Ltd., Evanston, I11. "l'Lee Pharmaceuticals, South El Monte, Calif. ~Beckman Model 979, Beckman Instruments, Inc., Fullerton, Calif. w Watford, England.
IOURNAL OF E N D O D O N T I C S I VOL 5, N O 9, SEPTEMBER 1979
!llnternationaI Scientific Instruments, Mountain View, Calif, 82 Super-ll, International Scientific Instruments, Mountain View, Calif. Drs. Wayman and Kopp were graduate students in endodontics during the investigations. Dr. Pinero is associate professor of histology, and Dr. Lazzar is professor of biochemistry, Dental Branch, University of Texas Health Science Center at Houston, 6516John Freeman Ave., Houston, 77025. Requests for reprints should be sent to Dr. Lazzari.
References 1. Callahan, J. Sulfuric acid for opening root canals. Dent Cosmos 36:957, 1894. 2. Harlan, A. Pulp digestion. Dent Cosmos 42:1272. 1900. 3. Auslander, W., and Roth, 14. Tryptar--its application in endodontics. Oral Surg 6:898-901, 1953. 4. Golden, S.S. and Musgrave, W.A. A clinical appraisal of Varidase in endodontics. Oral Surg 7:658-661, 1954. 5. Coolidge, E. Studies of germicides for treatment of root canals. JADA 16:698-712, 1929. 6. Ross, lt.J. Azochloramid in ro~t canal antisepsis. JADA 22:637-646, 1935. 7. 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(2):49-53, 1977. 8. Schilder, H., and Amsterdam, M. Inflammatory potential of root canal medicaments. A preliminary report including nonspecific drugs. Oral Surg 12:211-221, 1959. 9. Becker, G.L.; Cohen, S.; and Borer, R. The sequelae of accidentally injecting sodium hypochlorite beyond the root apex. Oral Surg 38(4):633-638, 1974. 10. Spangberg, L.; Engstr6m, B; and
I,angeland, K. Biologic effects of dental materials. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg 36:856-871, 1973. 11. Bence, R.; Madonia, J.V.; Weine, F.S.; and Smulson, M.A. A microbiologic evaluation ofendodontic instrumentation in pulpless teeth. Oral Surg 35:676-683, 1973. 12. Martin, H. Quantitative bactericidal effectiveness of an old and a new endodontic irrigant. J Endod 1(5):164-167, 1975. 13. Blechman, H., and Cohen, M. Use of aqueous urea solution in the field of endodontia: preliminary report. J Dent Res 30:503504, 1951. 14. Stewart, G.G.; Cobe, H.M.; and Rappaport, H. A study of a new medicament in the chemomechanical preparation of infected runt canals. JADA 63:33-37, 1961. 15. Goldberg, F., and Abramovich, A. Analysis of the effect of EDTAC on the dentina[ walls of the root canal. J Endod 3(3):101-105, I977. 16. von der Fehr, F.R. and Nygaard Ostby, B. Effect of EDTAC and sulfuric acid on root canal dentin." Oral Surg 16:199-205, 1963. 17. McComb, D.; Smith, D.C.; and Beagrie, G.S. The results of in vivo endodontic chemomechanical instrumentation-a scanning electron microscope study. J Br Endod Soc 9(1):11-18, 1976. 18. Heling, B.; Shapiro, S.; and Sciaky, I. An in vitro comparison of the amount of calcium removed by the disodium salt EDTA and hydrochloric acid during endodontic procedures. Oral Surg 19:531-533, 1965. 19. Loel, D.A. Use of an acid cleanser in endodontic therapy. JADA 90(1):148-151, 1975. 20. Woessner, J.E. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Binphy 93:440, 1961. 21. Stewart, G. Determination of the
approximate volumes of medication used in endodontic treatment. J Dent Res 27:24-26, 1948. 22. Gray, J.M., and Gallwas, G.E. Atomic absorption determination of serum calcium. Flame Notes 2:1, 1967. 23. Hughston, II.H.; Earle, L.S., and BinkIcy, F. Amino acid composition of proteins of human dentin. J Dent Res 38:323-327, 1959. 24. Weinreb, M.M., and Meier, E. The relative efficiency of EDTA, sulfuric acid, and mechanical instrumentation in the enlargement of rc• canals. Oral Surg 19:247-252, 1965. 25. Bell, G.H.; Davidson, J.N.; and EnslieSmith, D. Textbook of physiology arid biochemistry. Baltimore, Williams & Wilkins Co., 1972, p 838. 26. Softer, M.D. Chelation therapy. Springfield, Ill, Charles C Thomas, 1964, p6. 27. Neuman, W.F., and Newman, M.W. The chemical dynamics of bone mineral. Chicago, University of Chicago Press, 1958, p 142. 28. Brown, L.R., and Wheatcroft, M.G. Effect nf the diffusion of microbial growth factors through tooth substance on production of carious lesions in vitro. J Dent Res 45:830837, 1966. 29. Marshall, F.J.; Ma~ler, M.; and Dute, H.I,. Effects of endodontic treatments on the permeability of root dentine. Oral Surg 13:203-223, 1960. 30. Cohen, S.; Stewart, G.G., and Laster, L.L. The effects of acids, alkalies, and chelating agents on dentine permeability. Oral Surg 29:631-634, 1970. 31. Fraser, J.G., and Laws, A.J. Chelating agents: their effect on the permeability of root canal dentin. Oral Surg 41(4):534-540, 1976. 32. Trepagnier, C.M.; Madden, R.M; and Lazzari, E.P. Quantitative study of sodium hypochlorite as an in vitro endndontie irrigant. J Endod 3(5):194-196, 1977.
265
Citric and lactic acids as root canal irrigants in vitro Blake E. W a y m a n , DDS, MS; William M. Kopp, DDS, MS; Gerald J. Pinero, PhD; and E. P. Lazzari, PhD, H o u s t o n
T h e efficacy of solutions of lactic acid, t h r e e c o n c e n t r a t i o n s of citric acid, s o d i u m h y p o c h l o r i t e , a n d p h y s i o l o g i c saline u s e d as root canal irrigants w a s e v a l u a t e d . W i t h u s e of extracted, s i n g l e - r o o t e d h u m a n teeth, the a m o u n t s of h y d r o x y p r o l i n e a n d c a l c i u m r e m o v e d b y these irrigants w e r e m e a s u r e d . T h e t r e a t e d teeth w e r e t h e n e x a m i n e d w i t h a s c a n n i n g e l e c t r o n m i c r o s c o p e . It w a s c o n c l u d e d t h a t a 10% s o l u t i o n of citric acid as a lubricant, f o l l o w e d b y a 2.5% s o l u t i o n of s o d i u m h y p o c h l o r i t e as a n irrigant, a n d t h e n a g a i n u s e of the citric acid solution, will p r o d u c e clean canal walls with p a t e n t d e n t i n a l tubules.
A 50% solution of sulfuric acid' was one of the first of the many agents that have been utilized as root canal irrigants. Proteolytic enzymes ~-4 and chlorine-releasing agenW '''~ have also been used. Sodium hypochlorite has been the most commonly used root canal irrigant since its introduction and is an effective solvent of organic tissue5 It is also an irritant to healthy tissue"-" and an effective germicidal agent. TI' Other irrigants such as potentiated 1,5 pentanedia[, '~ urea peroxide, ':~ Gly-oxide," and hydrogen peroxide have been used. Ethylenediaminotetraacetic acid (EDTA) was the first reported chelating agent to be used as a root canal irrigant. '~-''; Concentrations of E D T A as small as 15% dissolve hydroxyapatite at least as well as a 20% solution of hydrochloric acid or a 50% solution of sulfuric acid.'" Several studies using a scanning electron microscope 1:'-'~'~ have shown that chelating agents consistently produced patent dentinal tubules
258
and, often, the cleanest canal walls. Another chelating agent, citric acid, has been recently reported by Loci'" to be an effective root canal irrigant when used alternately with sodium hypochlorite. Because such chelating agents as lactic and citric acids occur naturally in the body, these solutions are thought to be more acceptable biologically than many irrigants that are commonly in use. The purpose of this study was to compare the effectiveness of solutions of lactic acid, various concentrations of citric acid, sodium hypochlorite, and physiologic saline (control) as organic and inorganic tissue solvents. Biochemical a,Csays were done and scanning electron micrographs were taken to assess the effects of the various irrigants on the canal walls.
M E T H O D S A N D MATERIALS Hydroxyproline assay The hydroxyproline assay in vitro used 120 permanent human single-
rooted teeth. Previous histories for these teeth were not available, but, generally, they were removed for orthodontic or periodontal reasons. The anatomical crowns were removed with an Isomet* low-speed saw, and each tooth was examined for the presence of an intact pulp. If no pulp tissue was observed, the tooth was discarded. Each canal was instrumented through the apex to a size 80 or 90 file, rinsed with 2 ml of de-ionized water, and stored in 100% humidity at 4 C until used. The root apex was sealed with wax, mounted on wax squares, and placed randomly in one of lhe six sample groups for irrigation. The six irrigant solutions used were 0.86% physiologic saline (control); 5.25% sodium hypochlorite (pH adjusted to 11.7); 50% citric acid, (w/v, p H 1.6, prepared in the laboratory); Epoxylite 9060t cavity cleaner (commercial solution of 50% citric acid); 50% lactic acid, (v/v, p H 1.5, prepared in the laboratory); and
JOURNAL OF ENDODONTICS I VOL 5, NO 9, SEPTEMBER 1979
5.25% sodium hypochlorite and laboratory-prepared 50% citric acid solution alternated in varying sequences. Each irrigant was tested on 20 teeth. After instrumentation, the irrigating solution was deposited in the canal to the level of the surface of the sectioned tooth, and was allowed to remain for five minutes. When solutions were alternated, each solution remained in the canal for 21/2 minutes. The solutions were removed from the canal using enough paper points to completely dry the canal surface. The paper points were washed three times with 2 ml of deionized water per wash, which was collected, slant frozen, and lyophilized. Then, 2 ml of 6N hydrochloric acid was added to each dry sample; these were capped and heated for three hours at 130 C. The hydrochloric acid hydrolysate was removed by flash evaporation; 3 ml of de-ionized water was added and removed in 1-ml increments to dissolve the dry sample. The samples were again slant frozen, lyophilized, and prepared for assay by adding 2 ml of de-ionized water. This was divided into two 1-ml samples, to which 1 ml of de-ionized water was added. A reagent blank and a hydroxyproline standard were determined for each group. The assay for hydroxyproline was done according to the method by Woessner,-'" whereas the method outlined by Stewart'-" was used to determine the volume of the canal. Calcium assay
Six groups of five single-rooted human teeth were prepared for the calcium assay in the same manner described for the hydroxyproline assay. The teeth of each group were filled with one of the following irri-
gant solutions, which were left in the canals for five minutes: 0.86% physiologic saline (control); 50% lactic acid (v/v, pH 1.5); 50% citric acid (w/v, pH 1.6); 25% citric acid (w/v, pH 1.6); 10% citric acid (w/v, pH 1.6); and 5.25% sodium hypochlorite (Clorox) with the pH adjusted to 11.7. The samples for the calcium assay were collected and washed in the same manner described for the hydroxyproline assay. The l-ml samples from groups 1 and 6 were diluted with 1 ml of a lanthanum oxide solution (1,200 ppm). Groups 2, 3, 4, and 5 were diluted with 1.6 ml of the lanthanum oxide solution for each 0.4 ml of sample, because of higher concentrations of calcium in these groups. The optical density for each sample of known and unknown concentration was determined with an atomic absorption apparatus.~ 2~ Electron microscope
Canals of teeth selected for examination with the scanning electron microscope were prepared in the manner previously described for the biochemical assays. Three teeth from each of the following seven groups of solutions were examined: 0.86% physiologic saline; 50% lactic acid; 50% citric acid; 25% citric acid; 10% citric acid; 5.25% sodium hypochlo-
rite; and 50% citric acid used as a lubricant during instrumentation. The irrigants remained in the canals for five minutes, and were removed using paper points. The canals were then rinsed with 10 ml of de-ionized water delivered by needle and syringe, and were sealed with boxing wax. The teeth were notched buccally and lingually with a no. 700 tapered fissure bur in a high-speed handpiece and then were split longitudinally. The samples were dried in increasing concentrations of ethanol and then in amyl acetate. Carbon dioxide served as the transitional solvent in the critical point-drying apparatus.w The specimens were mounted on studs with a conductive adhesive and then were coated with a 500 A layer of gold in a sputter coater.II Photomicrographs of typical areas were taken at magnifications of 60 to 1,500 diameters. 82 The canals were evaluated for the appearance of debris, patent dentinal tubules, and residual pulp tissue. RESULTS The control group in the assay for hydroxyproline was tested with a physiologic saline solution irrigant that yielded no measural~le quantity of hydroxyproline (Table 1). The control irrigant yielded an average of
Table I. Comparison of all groups for hydroxyproline.
Sample group
Z,#g
~ #g
2 vol
Saline 5.2% hypochlorite 50% citric acid 50% lactic acid Epoxylite Combination-Varied Sequence hypochlorite/citric acid citric acid/hypochlorite
0.00 112.34 16.29 10.12 13.68 85.68
0.00 2.80 0.40 0.25 0.34 2.14
20.74 21.03 19.15 14.38 20.92 18.19
~/~g//zl 0.000 0.410 0.021 0.020 0.016 0.122
SD/~g/p.l 0.000 0.076 0.025 0.021 0.021 0.062
40.70 44.98
2.04 2.24
18.32 18.07
0.127 0.117
0.068 0.056 259
J O U R N A L OF E N D O D O N T I C S
Table 2 9 Comparison
Sample group 1.11 /~g//~l of calcium as determined from a 1-ml sample (Table 2). Scanning electron micrographs of the canals treated with saline solution disclosed a smeared layer probably consisting of organic and inorganic debris with no readily visible dentinal tubules (Fig 1). The sample group irrigated with the 5.25% solution of sodium hypochlorite yielded the greatest amount of hydroxyproline (0.14/~g/ ~1), which was significantly greater (P < .001) than all groups except the combination group, which yielded 0.12 /zg//fl (Table 1), The calcium assay for sodium hypochlorite gave a mean of 1.08 /~g/~l (Table 2). The scanning electron micrographs showed canal walls with a smeared layer of material (Fig 2) similar in appearance to that produced by physiologic saline solution. Some dentinal tubules were visible, but, in general, the instrumented canal surface was rough and irregular. In an uninstrumented area (Fig 3) globular dentin with patent dentinal tubules was seen, with apparently no organic matter present. The hydroxyproline assay for the group treated with 50% lactic acid solution produced results that were significantly different from the control (P < .001) with a mean of 0.020 ftg/~l (Table 1). In the calcium assay, lactic acid removed a mean amount of 8,82 ftg/~tl, which is also highly significant ( P < .001) when compared with the saline control. Scanning electron micrographs showed clean canal walls, but the dentinal tubules did not appear completely patent (Fig 4). The 50% citric acid solution prepared in the laboratory and the preparation of epoxylite 9060 yielded 0.021 and 0.016 /~g/btl of hydroxy260
Saline 50% lactic acid 50% citric acid 25% citric acid 10% citric acid Sodium hypochlorite ~Five
teeth per
group
I
V O L 5, N O 9, SEPTEMBER 1979
o f all g r o u p s f o r c a l c i u m . *
Y.~g
X/a,g
~ vol
,~ ~g//,tl
S.D. /.tg//d
101.96 749.02 970.50 708.88 697.20 90.03
20.39 149.80 194.10 141.78 139.44 I0.01
17.92 17.33 21.79 14.57 20.15 17.22
1.11 8.82 8.92 10.17 7.82 1.08
0.53 0.56 0.68 1,63 2.92 0.34
per five minute.
.
i,i
...
~
".
(~
F(r l-Instrumented root canal irrigated with physiologzc saline; canal appears oh'an, but ,o patent dentinal tubules are uisible. Some gross debris ('D ) is present.from splitting lechnique (o~g mag X 1,250).
t d, t
i
.
r
. '
9
.;r
5
.
9
,i
"~ ..
J
: *..
.
'
i
. ~,
t.. .
Fzig 2-Canal treated with sodium /?rpochlorile. Instrumented area shows no organtc debris. Rough. smeared appearance is thoueht to be tnot~amc material resulting from inslrU mentation, l)entinal tubules are z'isible, Ira/ occluded (orig mag x 1,500). ,, ~
.t~
. . ~.,r
. ,..
:...t, .
JOURNAL OF ENDODONTICS I VOL 5, NO 9, SEPTEMBER 1979
Fzg 5--Canal treated with 10% solution of citric acid. A rea shown is about mid-root and has eroded appearance seeming to e.~pose dentinal matrix.fiber (arrow). Dentinal tubules are patent and canal waU appears clean. Some debris (D) is present (ortg mag • 1,500).
'
Fig 3--Uninstrumented area of canal treated with sodium hypochlorite in which dentinal tubules are visible and no organic tissue can be distinguished. Irregular surface (G) is thought to be inorganic globular dentin (orig rnag X 1,500).
c
(
~
"
i~ ' ~
,,
{ t'
FtIr 4-Canal treated with lactic acid appears clean with obvious dentinal tubules, some of which are open. whereas others appear occluded (orzir mag • 1,250).
.h
~
.
l~'ti~ 6--Canal treated with 5(P/~ solution of citric acid. Dentinal tubules (7") are wide open and tooth su!filce is smooth and clean. Area has been instrumented (orzg mag • 1,500).
).
4..,
k ."N .
.
t"i ,
; a..,
proline (Table 1), respectively, with no statistically significant difference. This is, however, significantly more (P < .001) than the control group. T h e calcium assay showed a highly significant difference (P < .001) for all three concentrations of citric acid when compared with the control. T h e mean for the group treated with a solution of 50% citric acid was 8.92/~g//~l; 10.17/ug//~l for the group treated with a 25% solution of citric acid, and 7.82 /ag//~l for the group that received a 10% solution of citric acid, (Table 2). Micrographs of the teeth irrigated with the three concen-
trations of citric acid showed canal walls that were generally free of the smeared appearance. There were more patent tubules with even a 10% concentration of citric acid (Fig 5) than with the sodium hypochlorite. The teeth treated with 50% citric acid (Fig 6) had dentinal tubules that were generally more patent, and canal walls that were cleaner, than those treated with the 10% and 25% concentrations. However, there was a great variation in the patency of the tubules at the lower concentrations. Figure 7, taken near the cementoenamel junction, shows that citric 261
JOURNAL OF ENDODONTICS I VOL 5, NO 9, SEPTEMBER 1979
acid had little effect on the organic matrix. The alternation of sodium hypochlorite and 50% citric acid solution yielded 0.12 ffg/ffl of hydroxyproline, which was significantly more (P < .100) than the amount yielded by the control (saline solution) or chelating agents, but less than that yielded by the group treated with 5.25% sodium hypochlorite solution. The sequence of application of the solution was varied, but there was no significant difference in the results of the assay relating to the sequence in which the agents were used. Scanning electron micrographs of the teeth in which the 50% solution of citric acid was used as both a lubricant and irrigant showed a relatively smooth, clean canal surface with patent dentinal tubules in the instrumented areas (Fig 8). In the uninstrumented areas, the dentinal tubules were patent, and cells, which may be odontoblasts and fibroblasts, were present (Fig 9).
Fzg 7-Canal treated with 50% solution of citric acid, near the cementoenamel junction in instrumented area. Undestrored fibers (F) remain. This is thought to be area in which calcification was possibly occurring. Note how fine matrix fibers have been exposed (arrow) (orzg mag X 1,500).
#J P
"4"'
..
DISCUSSION Analysis of amino acids in dentin protein has shown that much more hydroxyproline is present in dentinal collagen than in other types of collagen. '-':~ Because collagen is a major component of dentin, the measurement of hydroxyproline removed from dentin is an ideal method of determining the effect of the irrigants on the dissolution of organic material of the dentin and pulp. The assay for hydroxyproline according to Woessner'-'" has been shown to be accurate and simple for small amounts of this amino acid. The canal volume of each of the teeth was different and would have given greater amounts of hydroxyproline for a larger canal. For this reason, the 262
,,a;. ~J .F~
.
~.~. t
,, ..j
"
.q r
.
Fig 9-Root canal in which 50% solution of citric acid'was used as lubricant. Uninstrumented area near CI:.J shows cellular fibrous components (C). Note clean, open dentinal tubules (7~) (orig mag x 800).
,,..r.
9
}
/'
b'zg 8--Root canal in which 50% solution of citric acid was used as lubricant. Canal su~lce appears clean with hzgh(r ~,isible patent dentinal tubules. Instrument marks (1) are present," no smeared layer is seen (ortlg mag • 1,400).
JOURNAL OF E N D O D O N T 1 C S
VOL 5, N O 9, SEPTEMBER 1979
COMPARISON OF ALL GROUPS FOR HYDROXYPROLINE ~- ug/ul 0.14 0.13 0.12
/ ,/jZp ///
0.11
H;':"; .'//.." /.../
0.10
.
'5,
.
'
..~
0.09 ,'//.
0.08
,
--,;.,>..
.
.
0.07
/'/:, I/:,
",,
0.06
>;/',,-:: ,/:
'
:q,;).,)
0.05 ' 0.04 '
"V,>#.;..
0.03 '
/,,>~
0.02
,////
."
.
.;;...;.
. ;
Fq~ lO-,'ffean calm's (y~,lff/) ?lhldro.wpr,dim' rcmoccd br z,ar/ou.s Jrtlgant.~ during.licr-mmuh' peri~M
,, : .;:"; i :
..;,.;..:
<'9 9:9149149149
1"' '".';i;;
0.01 ( ,/i.
PHYSIOLOGIC
50%
50%
EPOXYLITE
SALINE
LACTIC ACiD
CITRIC ACID
9060
/r
SODIUM
COMBINATION-
HYPDCHLORITE VARIED SEQUENCE
COMPARISON OF ALL GROUPS FOR CALCIUM ug/ul 11 10
",..;;
9 '
!'/ / /
,.J
/;
/
/
I
/ ' , "1
8 7
"/
6
k'(Z" 11 Mean r (ffgltd) qf calcium remot,ed by cartous irr(gants during rice-minute per,od.
9
J
,'/
;"/..y'/2~
//./...//.
5 4
/. . . . . . . /./'...
-,.///
3
///
2
.,/...
""'~--//;"""1
-q
)..-,
' : Z ' i . / .. .
.,.-
I
volumes of the canals were determ i n e d a c c o r d i n g to the m e t h o d of Stewart. :~ In the h y d r o x y p r o l i n e study, the physiologic saline did not remove a m e a s u r a b l e a m o u n t , a n d it was shown t h a t the effect o f the 50% solutions of citric a n d lactic acid on the dissolution of organic tissue is also m i n i m a l . However, the h y d r o x y proline dissolved by the sodium hypochlorite was seven times more than the a m o u n t r e m o v e d by the chelating agents (Fig 10). In the calcium analysis, lactic acid and the three concentrations of citric acid removed seven to nine times as
;1
9 ;. • <; %.
;, ,,. . .:,.. .:,..,. .....
PHYSIOLOGIC
50%
50%
26%
10%
SALINE
LACTIC ACID
CITRIC ACID
CITRIC ACID
CITRIC ACID
much c a l c i u m as the physiologic saline control and the sodium h y p o c h l o r i t e (Fig 11). T h e 25% solution of citric acid removed slightly more c a l c i u m than the other chelating solutions tested. However, this value was not statistically significant (P < .05), a n d the a p p a r e n t increase is t h o u g h t to be due to the small n u m b e r of teeth tested in each group. T h e statistical analysis indicates a considerable deviation from the mean in m a n y of the groups, p e r h a p s because of the wide range of patients from w h o m these teeth were obtained. T h e results of the h y d r o x y p r o l i n e
G:;:~
SODIUM
HYPOCHLORITE
a n d calcium assays indicate that sodium h y p o c h l o r i t c is a much better solvent of organic tissue c o m p o n e n t s than are the c h e l a t i n g agents, citric acid a n d lactic acid. IInwcver, sodiu m hypochlorite has litth" effect on the inorganic m a t e r i a l tbund within the dentin 9 Scanning electron m i c r o g r a p h s showed that all concentrations of citric and lactic acid p r o d u c e d much cleaner canal walls and more p a t e n t d e n t i n a l tubules than the saline control or sodium hypochlorite. T h e d e n t i n a l particles seen in all the groups e x a m i n e d are thought to result from the techniques used in 263
JOURNAL OF E N D O D O N T 1C S I VOL 5, N O 9, SEPTEMBER 1979
micrographs studies are consistent with the results of the biochemical tests done in this study. In contrast, the micrographs of the teeth treated with sodium hypochlorite (Fig 2, 3) show no fibrous or cellular tissue and indicate that the solvent action of this irrigant is on the organic tissue of the root canal. The ideal irrigant is thought to be one that is germicidal, dissolves organic and inorganic tissue, and can be quickly metabolized with no adverse effect should it be accidently expressed beyond the apex. The germicidal effect of sodium hypochlorite has been well established, and its ability to dissolve organic tissue and cellular debris in the root canal has also been demonstrated7 The irritation to normal tissues by sodium hypochlorite, however, remains a concern. ~-'' Citric and lactic acid occur naturally in the body. Citric acid is found in mitochondria '-':' and is used by blood banks to prevent coagulation.'-'" Citric acid has also been shown to be the most effective acid in altering the solubility of hydroxyapatite. '-'~ Lactic acid occurs in the muscles under anaerobic conditions during vigorous muscular exerciseP :' Dentinal permeability to microorganisms is also of concern to the endodontist; 1''-'~ open dentinal tubules are thought to allow intracanal medicaments to enter and destroy any bacteria that may be present, '-'~:~~ Some investigators have found that chelating agents increase the dentinal permeability,'" whereas others reported a reduction.:" The results of this and another study :~: suggest that the use of two irrigants would yield the best results. Initially, a 10% solution of citric acid as a lubricant and hydroxyapatite solvent should be used, which helps 264
to maintain patent dentinal tubules. This should be followed by a 2.5% solution of sodium hypochlorite applied for five minutes :l'-'with frequent changes to dissolve the remaining organic material and destroy any microorganisms that may be present. Further instrumentation with a 10% solution of citric acid is recommended to ensure that the canal walls are clean and that the dentinal tubules are patent. It is believed that the use of a 10% solution of citric acid and a 2.5% solution of sodium hypochlorite, as described, removes almost all organic and inorganic debris from the canal. The scanning electron micrographs indicate that relatively germfree and clean canals with patent dentinal tubules are produced. This may be important clinically in allowing intracanal medicaments to be more effective and in permitting better obturation with the filling materials available. SUMMARY Concentrations of 10%, 25%, and 50% citric acid and 50% lactic acid were used as root canal irrigants to determine their effectiveness as tissue solvents in vitro. These solutions were compared with solutions of sodium hypochlorite and a saline control. The hydroxyproline values were used to indicate the solvent action of the irrigating solutions on the organic components of the tooth. Results show that the 5.25% sodium hypochlorite solution produced seven times more hydroxyproline than the preparations of citric acid or lactic acid, indicating that sodium hypochlorite produces a significantly greater ( P < .001) dissolution of organic tissue. The calcium values determined for all test agents indi-
cated the effect of these solutions on the inorganic dentinal components. Results showed that the solutions of citric and lactic acid all dissolved seven to nine times as much calcium as the sodium hypochiorite and the saline control, and this difference was f o u n d to be significant (P < .001). The solutions of citric and lactic acid were thought to act as chelating agents. The effects of the various irrigants on surfaces of the root canals were evaluated with the scanning electron microscope. All solutions of citric and lactic acid used only as an irrigant after dry instrumentation produced cleaner, smoother walls than did the solutions of sodium hypochlorite or the saline control. The 10% concentration of citric acid opened up the dentinal tubules and cleaned the canal wall, but not as effectively as the stronger concentrations of citric acid, although it was equal in the ability to dissolve minerals. Lactic acid left a film on the surface, possibly because of its greater viscosity and incomplete removal. Combined with instrumentation, the citric acid produced clean canal walls and patent dentinal tubules. The sodium hypochlorite produced a cleaner canal surface than did the saline control, but did not remove the debris from the dentinal tubules that resulted from the instrumentation of the root canal. The patent tubules and clean canal walls may be desirable clinically in allowing greater penetration of intracanal medicaments or filling materials into the dentinal tubules.
*Buehler Ltd., Evanston, I11. "l'Lee Pharmaceuticals, South El Monte, Calif. ~Beckman Model 979, Beckman Instruments, Inc., Fullerton, Calif. w Watford, England.
IOURNAL OF E N D O D O N T I C S I VOL 5, N O 9, SEPTEMBER 1979
!llnternationaI Scientific Instruments, Mountain View, Calif, 82 Super-ll, International Scientific Instruments, Mountain View, Calif. Drs. Wayman and Kopp were graduate students in endodontics during the investigations. Dr. Pinero is associate professor of histology, and Dr. Lazzar is professor of biochemistry, Dental Branch, University of Texas Health Science Center at Houston, 6516John Freeman Ave., Houston, 77025. Requests for reprints should be sent to Dr. Lazzari.
References 1. Callahan, J. Sulfuric acid for opening root canals. Dent Cosmos 36:957, 1894. 2. Harlan, A. Pulp digestion. Dent Cosmos 42:1272. 1900. 3. Auslander, W., and Roth, 14. Tryptar--its application in endodontics. Oral Surg 6:898-901, 1953. 4. Golden, S.S. and Musgrave, W.A. A clinical appraisal of Varidase in endodontics. Oral Surg 7:658-661, 1954. 5. Coolidge, E. Studies of germicides for treatment of root canals. JADA 16:698-712, 1929. 6. Ross, lt.J. Azochloramid in ro~t canal antisepsis. JADA 22:637-646, 1935. 7. 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(2):49-53, 1977. 8. Schilder, H., and Amsterdam, M. Inflammatory potential of root canal medicaments. A preliminary report including nonspecific drugs. Oral Surg 12:211-221, 1959. 9. Becker, G.L.; Cohen, S.; and Borer, R. The sequelae of accidentally injecting sodium hypochlorite beyond the root apex. Oral Surg 38(4):633-638, 1974. 10. Spangberg, L.; Engstr6m, B; and
I,angeland, K. Biologic effects of dental materials. Toxicity and antimicrobial effect of endodontic antiseptics in vitro. Oral Surg 36:856-871, 1973. 11. Bence, R.; Madonia, J.V.; Weine, F.S.; and Smulson, M.A. A microbiologic evaluation ofendodontic instrumentation in pulpless teeth. Oral Surg 35:676-683, 1973. 12. Martin, H. Quantitative bactericidal effectiveness of an old and a new endodontic irrigant. J Endod 1(5):164-167, 1975. 13. Blechman, H., and Cohen, M. Use of aqueous urea solution in the field of endodontia: preliminary report. J Dent Res 30:503504, 1951. 14. Stewart, G.G.; Cobe, H.M.; and Rappaport, H. A study of a new medicament in the chemomechanical preparation of infected runt canals. JADA 63:33-37, 1961. 15. Goldberg, F., and Abramovich, A. Analysis of the effect of EDTAC on the dentina[ walls of the root canal. J Endod 3(3):101-105, I977. 16. von der Fehr, F.R. and Nygaard Ostby, B. Effect of EDTAC and sulfuric acid on root canal dentin." Oral Surg 16:199-205, 1963. 17. McComb, D.; Smith, D.C.; and Beagrie, G.S. The results of in vivo endodontic chemomechanical instrumentation-a scanning electron microscope study. J Br Endod Soc 9(1):11-18, 1976. 18. Heling, B.; Shapiro, S.; and Sciaky, I. An in vitro comparison of the amount of calcium removed by the disodium salt EDTA and hydrochloric acid during endodontic procedures. Oral Surg 19:531-533, 1965. 19. Loel, D.A. Use of an acid cleanser in endodontic therapy. JADA 90(1):148-151, 1975. 20. Woessner, J.E. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Binphy 93:440, 1961. 21. Stewart, G. Determination of the
approximate volumes of medication used in endodontic treatment. J Dent Res 27:24-26, 1948. 22. Gray, J.M., and Gallwas, G.E. Atomic absorption determination of serum calcium. Flame Notes 2:1, 1967. 23. Hughston, II.H.; Earle, L.S., and BinkIcy, F. Amino acid composition of proteins of human dentin. J Dent Res 38:323-327, 1959. 24. Weinreb, M.M., and Meier, E. The relative efficiency of EDTA, sulfuric acid, and mechanical instrumentation in the enlargement of rc• canals. Oral Surg 19:247-252, 1965. 25. Bell, G.H.; Davidson, J.N.; and EnslieSmith, D. Textbook of physiology arid biochemistry. Baltimore, Williams & Wilkins Co., 1972, p 838. 26. Softer, M.D. Chelation therapy. Springfield, Ill, Charles C Thomas, 1964, p6. 27. Neuman, W.F., and Newman, M.W. The chemical dynamics of bone mineral. Chicago, University of Chicago Press, 1958, p 142. 28. Brown, L.R., and Wheatcroft, M.G. Effect nf the diffusion of microbial growth factors through tooth substance on production of carious lesions in vitro. J Dent Res 45:830837, 1966. 29. Marshall, F.J.; Ma~ler, M.; and Dute, H.I,. Effects of endodontic treatments on the permeability of root dentine. Oral Surg 13:203-223, 1960. 30. Cohen, S.; Stewart, G.G., and Laster, L.L. The effects of acids, alkalies, and chelating agents on dentine permeability. Oral Surg 29:631-634, 1970. 31. Fraser, J.G., and Laws, A.J. Chelating agents: their effect on the permeability of root canal dentin. Oral Surg 41(4):534-540, 1976. 32. Trepagnier, C.M.; Madden, R.M; and Lazzari, E.P. Quantitative study of sodium hypochlorite as an in vitro endndontie irrigant. J Endod 3(5):194-196, 1977.
265