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Pulp capping in monkeys with a calcium-hydroxide compound, an antibiotic, and a polycarboxylate cement
Pulp capping in monkeys with a calcium-hydroxide compound, an antibiotic, and a polycarboxylate cement George X. McWalter, Commander (DC) USN, Castro Valley, Calif., Abdel H. El-Kafrawy, B.D.S., M.S.D.,” and David 3”. Mitchell, D.D.S., Ph.D.,+* Indianapolis, Ind. INDIANA
UNIVERSITY
SCHOOL
OF DENTISTRY
In this histologic study, the pulps of seventy-four permanent teeth of four monkeys were mechanically exposed, contaminated with saliva, and capped. The teeth were extracted at 15, 50, 100, and 200 days. Of the teeth treated with Keflin (an antibiotic), only 8 per cent reacted satisfactorily. A calcium-hydroxide compound gave a 96 per cent satisfactory response. Combining only the lo&day and 200-day teeth, a polycarboxylate cement gave a 90 per cent success figure.
T
here is a widespread belief among dentists that, when pulpitis due to pulp exposure develops, the treatment indicated is ideally root canal therapy or, if this is not available, extraction. A problem with this attitude is that, for various reasons, root canal therapy is frequently not done. It is time consuming, it demands special skills, and, therefore, for many patients it becomes economically unfeasible. This is one reason for the estimated 250,000 or more teeth that are extracted per day in the United States.l Studies have shown that pulpitis may be reversible,2-* and, therefore, may be treated conservatively. If vital-pulp therapy fails, a choice can then be From a thesis submitted by the senior author to the faculty of Indiana University School of Dentistry in partial fulfillment of the requirements for the M.S.D. degree. The opinions and assertions contained herein are those of the writers and are not construed as official or as reflecting the views of the Navy Department or the naval servi large. *Assistant Professor, Department of Oral Diagnosis/Oral Medicine. **Professor and Chairman, Department of Oral Diagnosis/Oral Medicine.
90
Volumk; 36 Numbei 1
Pulp capping
in monkeys
91
Fig. 2. Photograph showing typical, mechanically exposed pulps.
made between exodontia and a root canal filling. The importance of this “vital” aspect of endodontic treatment cannot be overemphasized. Although innumerable pulp-capping agents have been advocated, the search for an ideal capping material has continued. The purpose of this investigation was to determine the reactions of contaminated pulps of permanent monkey teeth to direct pulp capping with a calcium-hydroxide compound, an antibiotic, and a polycarboxylate cement. Calcium hydroxide, usually in pure powder form mixed with some vehicle, has been the most popular of the pulp-capping agents. Since microorganisms seem to be a major factor in the eventual death of the p~lp,~ the rationale for including an antibiotic in the present study is obvious. Polycarboxylate cements have shown great promise in that they are claimed to have all the attributes of the two most popular cements in dentistry (zinc oxide and eugenol and zinc phosphate cement) without their disadvantages.6 A polycarboxylate cement was found to be almost nonirritating when implanted subcutaneously in rats or placed in deep cavities in monkey teeth.7 The use of this material as a pulp..I capping agent puts it to an extreme biologic test. METHODS AND MATERIALS Four young monkeys were used-a Macaca nemestrina, a Macaca speciosa, and two Macaca mulatta. The animals were tranquilized by intramuscular injection of 5 mg. of phencyclidine hydrochloride. * Anesthesia was then obtained by intravenous injection of 22 mg. of sodium pentobarbitalf per kilogram of body weight. Additional amounts were injected as needed. A full-mouth set of radiographs was made with the exposure settings of 60 KVP, 6 Ma., and 0.25 second. An increased vertical angulation was used to conform to the monkey’s jaw. Seventy-four pulp exposures were made in twenty-two anterior and fifty-two “Sernylan, Bio-ceutic Laboratories, Inc., St. Joseph, MO. tSomnopenty1, Pitman-Moore, Inc., Fort Washington, Pa.
Oral Surg. .July. 1973
posterior teeth. This included every pcrmancnt tooth present cxc*ept t,hc c~lisl)ids. A%class v preparation (&‘ig. 1) was made on the facial surfac? CJf ea(‘11 toot11 with an air-driven, No. 701, tapcretl fissure carbide bur at high spoctl untl~r a watc>r coolant. Short blasts of air lverc used to remove debris. A ~11 was made in the center of the cavity floor with a slowly rotating Xo. 4 round carbide bur until pink dentin indicated that an exposure was imminent. An explorer point was then used to make the exposure, entering the pulps approximately 1 mm. This careful procedure was used to minimize the number of dentin chips introduced into the pulp. An attempt was made to keep the diameter of the exposure about 1 mm. On a few occasions the exposure was inadvertently made with the bur. No aseptic procedures were used since contamination of the pulp was intentional. A water spray was used to clean dentin from the cavities, and saliva and materia alba, if present, were introduced into the exposure to contaminate the pulp. The pulps were left open to the oral environment for 3 to 51/, hours before the pulp capping was done. Then each cavity was cleansed with a spoon excavator and water spray. Usually, hemorrhage was induced by this procedure, and cotton pellets were used to control it. The three compounds used as the pulp-capping agents were a caIcium hydroxide compound (Dyeal”), a polycarboxylate cement (Durelonf ), and the antibiotic, sodium cephalothin (Keflint). Because each has a distinctive appearance, it was impossible not to know which agent was being used. Each agent was used in every third tooth available according to t,he l-32 numbering system for teeth. Both Dycal and Durelon were mixed on a 1:l base-liquid ratio. A layer of 0.5 mm. or more was left against the pulp. The Keflin powder was made into a paste with the use of sterile water. This was deposited on the pulp and a piece of gold foil covering all of the cavity floor was placed to lend support during the condensation of amalgam. After a layer of Copalite varnish was placed in the cavity covering the margins, an 8:5 mercury-alloy ratio of amalgam was inserted. The animals were maintained on a normal laboratory diet.5 Each monkey received at least one intraperitoneal injection of Procion Brilliant Red H-SBS,ll an in vivo hard-tissue marking agent. The dosage was 100 mg. per kilogram of body weight. The dye was used to determine the amount of reparative dentin deposited between the time of injection and the time of extraction. One monkey was injected 8 days after pulp capping, the second 30 days later, the third at 60 and 90 days, and the fourth 150 days after capping. The teeth were removed at intervals of 15, 50, 100, and 200 days after pulp capping. On the day of extraction, the oral cavity was examined and full-mouth
*L. D. Caulk Co., Milford, Del. tPremier Dental Products Co., Philadelphia, Pa. $Eli Lilly and Co., Indianapolis, Ind. $Monkey Chow, Ralston-Purina Co., St. Louis, MO. III. C. I., Organ&, Inc., Providence, R.I.
Volume 36 Number 1
Pulp capping
in monkeys
93
Fig. 8. Photomicrograph of a Keflin-treated pulp at 15 days. Notice the severe inflammation with abscess formation. (Hematoxylin and eosin stain. Original magnification, x35.)
radiographs were made. Alveolar bone on the facial surface was removed first with a mallet and chisel. Elevators and forceps were used to deliver the teeth carefully in order to avoid root fracture. Immediately after extraction, 3 mm. of the apical portions of the roots was’cut off with a bur rotated at high speed under a water spray. The specimens were then placed in 10 per cent formalin until ground down on one proximal surface to ensure adequate pulp fixation. Decalcification was accomplished with 5 per cent formic acid. Serial 7-pm thick paraffin sections were then cut through the extent of the cavity, including the exposure site. Alternate slides were stained with hematoxylin and eosin. Unstained sections were examined by fluorescent microscopy for Procion red markings. Selected sections were stained by the Brown and Brenn method to demonstrate the presence of bacteria in the pulps. Eight untreated teeth were also included in the study for control purposes. RESULTS Of the three materials, Durelon was the most easily delineated on the radiographs since it was distinctly radiopaque. Dycal was only slightly more radiopaque than the dentin. Keflin, since it was covered with gold foil, could not be distinguished under the alloys, Gross internal resorption was observed in two teeth capped with Keflin, one for 100 days and the other for 200 days. A radiolucency suggestive of external resorption appeared at the apex of another loo-day, Keflin-treated tooth. Otherwise, no significant roentgenographic abnormalities were noted. The microscopic findings are summarized in Table I. Pulp reactions to the capping agents were classified as satisfactory or unsatisfactory. A satisfactory response demonstrated complete absence of inflammation within the pulp or a scattering of a few inflammatory cells localized at the exposure site, with or without complete bridging. All pulps that demonstrated a moderate inflammatory response at the exposure site or severe inflammation with abscess
94
HcWalter,
Table
El-Knfmwy,
Oral Surg. July, 1973
md Mitchell
I. Summary of the microscopic findings
in seventy-four
permanent teeth capped
Kejlin
Tooth No.
Calcifio briaghg Inflammation Capped 15 days (Mg:;reM1, 21 teeth) 4 0” 8 Severe 12 Severe 0” 14 Severe 0 20 Severe 26 Severe t 31 Severe 0 Cappei 50 days (Mgt~feM6, 23 teeth) 4 10 14 2
Mild i Severe 0 Moderate 0 Severe Severe i 26 Severe Moderate ?I Cap&i 100 days (z$ey M5, 18 teeth) 7 Incomplete 10 Severe 15 Severe 0” 29 Severe 23 i 30 Severe 0 Cappei 200 days (M%;:;z M2, 12 teeth) 1’9”
Severe Severe Severe
28 *O = Absent. t- = Unsatisfactory. t+ = Satisfactory.
:: i
Pulp
response
Tooth No.
Inflammation
10 15 19 25 30
Moderate Severe Mild Moderate Severe Mild Moderate
-t
+t
f
5 8 12 15 20 24 28 31
Mild
0 0
Mild Moderate 0 Mild Moderate
2 8 13 18 24 26 1: 21 29
0 Mild Moderate Mild
formation or pulp necrosis were regarded as having responded unsatisfactorily to the capping agent. At 15 days, all of the seven Keflin-treated pulps demonstrated severe inflammatory reactions with abscesses (Fig. 2). At 50 days, only one of eight teeth similarly treated gave a satisfactory reaction. Six teeth were capped with Keflin for 100 days. Of these, only one gave a satisfactory response, two demonstrated severe inflammation with abscess formation, and three displayed complete necrosis. Multinucleated giant cells in association with internal resorption were observed in three of the Keflintreated teeth. At 200 days, all four of the Keflin-treated pulps demonstrated severe inflammation with abscesses. In one tooth, gross internal resorption had penetrated to the periodontal ligament. At 15 days, only two of seven pulps capped with Durelon responded satisfactorily. These showed a scattering of a few inflammatory cells at the exposure site. Three pulps showed inflammation of a moderate intensity, and the two other pulps showed severe inflammation with abscess formation. Of eight teeth capped with Durelon for 50 days, 6 responded satisfactorily. Of these, three demonstrated no inflammation, and three showed a few in-
Volume 36 Number L
with Keflin,
Durelon,
Pulp
capping
in monkeys
95
and Dycal
Dwebn
DYCQJ
Caloi$o bridgildg
Pulp response
Tooth No.
+
2 5 9 :i
Mild Mild Mild Mild Severe
+
29 24
Mild 0
0 0
Incomplete Incomplete Incomplete Incomplete 0 Incomplete i Incomplete Incomplete Incomplete Complete Complete Incomplete Incomplete Incomplete 0 Incomplete
2
Caloi$o
I I@ammatio*
bridging Incomplete . 0 0 Incomplete 8 0
i; 29
Complete Complete Complete Complete Complete Complete Complete
i 14 19 25 31
Complete Complete Complete Complete Complete Complete
i 13
4 ii’ 30
Pulp I response + + +
Complete Complete Complete Complete
flammatory cells at the exposure site. Incomplete bridging was evident in five instances. Usually, no true dentin bridge appeared to occur at the site of the exposure, but dentin walls adjacent to the exposure and dentin chips appeared to be the main nidus for bridging (Fig. 3). When no inflammation was present, the usual picture at the exposure site was a compressed zone of fibroblasts, collagen fibers, and some capillaries. At 100 days, all six Durelon-capped pulps showed bridging and no inflammation (Fig. 4). A few times when a bridge appeared to be complete, careful study of serial sections revealed some vital tissue penetrating through the calcified material. As in the 50-day specimens, dentin chips and adjacent pulpal walls seemed to be the main stimuli for dentinogenesis. Typically, between the Durelon and the bridge were necrotic debris, degenerated red blood cells, and occasionally dentin chips and vital tissue. At 200 days, one of the four Durelon-capped pulps had moderate inflammation. The integrity of the amalgam seal was suspect in this case since the amalgam fell out during extraction. Reaction in the three other pulps was considered to be satisfactory: two showed a few inflammatory cells at the exposure site, and the other pulp was free of inflammation. All three pulps showed incomplete bridging.
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and Mitchell
Oral Surg. July, 1973
Fig. 8. An example of no inflammation in a Durelon-treated pulp at 50 days. Some vital tissue is present on the cavity preparation side. Examination of the serial sections showed the dentin bridge to be incomplete. (Hemntoxylin and eosin stain. Original magnification, x35.) Fig. 4. Microscopic section of a Durelon-capped pulp at 100 days with no inflammation. Exceptionally, this bridge was complete. Some tissue debris is present on exposure side of the dentin bridge. (Hematoxylin and eosin stain. Original magnification, x35.)
Six of the seven teeth capped with Dycal for 15 days gave satisfactory responses. Two of them exhibited no inflammation, and four showed very mild inflammation. Usually, some Dycal was present either in the preparation area or in small amounts in the pulpal tissue within macrophages (Fig. 5). The Dycal was frequently observed in direct contact with vital tissue (Fig. 6). At other times, necrotic debris, including extravasated red blood cells, was seen under the pulp-capping material. One specimen capped with Dycal for 15 days showed severe inflammation with abscess formation. At 50, 100, and 200 days, all seventeen pulps capped with Dycal were free of inflammatory cells. Each had a complete dentin bridge (Figs. 7 and 8). The calcified material that first formed was usually amorphous with cellular inclusions; then irregular tubular dentin was present, and, finally, dentin of regular tubular structure appeared. Three sources of dentinogenesis were observed : from the dentinal walls near the exposure site; around dentin chips; and at the exposure site in contact with the Dycal (Fig. 7). Sections from twenty teeth were selected for a modified Brown and Brenn stain, All of the Keflin sections were negative for bacteria, even a pulp with complete necrosis. The only Dycal-treated pulp that demonstrated any bacteria was the one with severe inflammation at the 15-day interval. Gram-positive
Volume Number
36 1
Pulp capping in molzlceys 97
Fig. 5. Photomicrograph of a Dycal-capped pulp at 15 days with no intlammation. Many dentin chips are present. Vital pulp tissue is in direct contact with Dycal. (Hematoxylin and eosin stain. Original magnification, x35.) Fig. 6. Higher magnification of exposure site in Fig. 5 showing Dycal (Mao76 at the top) in direct contact with vital pulp tissue. (Hematoxylin and eosin stain. Original magnification, x250.)
bacteria were demonstrated in four of the Durelon-treated pulps, which also were inflamed to varying degrees. Gram-negative organisms were either absent or too difficult to identify. No microorganisms were observed in teeth without pulpal inflammation. The monkey that provided the E-day specimens was injected with Procion red 8 days after pulp capping. No specimens showed labeling. Dye was administered 30 days after capping to the animal that supplied the 50-day teeth. Good labeling of the Dycal-treated teeth was observed. The continuous fluorescent line in the calcified bridge indicated that a complete bridge was present at approximately 30 days after operation. The monkey supplying the KM-day teeth was injected with the dye at 60
98
MeWalter,
El-Kafrawy,
and Mitchell
Oral Surg. July, 1973
Fig. 7. An example of a complete dentin bridge and no inflammation in a Dycal-capped pulp at 100 days. Some Dyeal is present in the pulp. (Hematoxylin and eosin stain. Original magnification, x35.) Fig. 8. A typical Dycal-treated pulp at 200 days with no inflammation. Dentin bridge is complete and the Dycal (black) abuts directly against the calcified bridge. Notice the reparative dentinogenesis on the dentinal walls near the exposure, around dentin chips and at the exposure site in contact with the Dycal. (Hematoxylin and eosin stain. Original magnification, x35.)
and 90 days after capping. Where much tertiary dentin had formed, the labeling was usually good. Two distinct lines were usually seen. The amount of reparative dentin formed between 60 and 90 days seemed to be diminishing. Interestingly, two lines were seen in the dentin walls of canals of nontreated teeth. Procion red was administered to the animal providing the 200-day specimens at 150 days after operation. No marking was seen in the Keflin-capped teeth. The lines were vary faint, if seen at all, in the Dycal-treated and Durelon-treated teeth. This indicated that reparative dentin formation had ceased or markedly diminished in rate by 150 days after capping. DISCUSSION
All pulps used in this study were contaminated 3 to 51/s hours with saliva or plaque before pulp capping, since contact of microbial flora with the pulp is a major factor in pulpitis and pulp necrosis .5 We speculated that a broad-spectrum antibiotic, such as Keflin, would kill the microorganisms and thereby enhance the healing of the pulp. However, of the twenty-five teeth treated with this antibiotic, only two reacted satisfactorily. In selected sections stained to demonstrate bacteria, none was found, even in a tooth with complete pulpal necrosis. We can assume that, even though Keflin killed the bacteria, it was so severely irritating to the pulp that the tissue was irreversibly damaged. The pH of an aqueous solution of Keflin is 5.2.8
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Pulp
capping in monkeys
99
In the Durelon-treated teeth, only two of seven pulps had satisfactory reactions at the 15-day interval. The success rate rose to 75 per cent at 50 days. When the lOO-day and 200-day specimens were combined, a 90 per cent success figure was reached. The amalgam seal was suspect in the one tooth in the latter category where moderate inflammation was observed. In two instances a complete dentin bridge formed across the exposure site under Durelon ; however, these were exceptions. Frequently, an incomplete bridge formed, with vital tissue between it and the exposure site. Dentin chips and the adjacent pulpal walls apparently acted as niduses which stimulated dentinogenesis, and sometimes coalescence of this newly formed dentin resulted in calcified bridging. Some necrotic debris was occasionally observed at the exposure site. This was probably due to necrosis caused by the trauma of the mechanical exposure and not to irritation from the Durelon. Only one of the twenty-four teeth treated with Dycal gave an unsatisfactory response, and this was at the 15-day interval. At 50 days and later, none of these teeth exhibited any inflammation and all demonstrated complete dentin bridges, confirmed by the study of serial sections and vital-dye markings. The pulpal reaction to Ca(OH), pastes typically leaves a necrotic zone between the bridge and the paste.9 This often was not the case with Dycal, at the intervals studied, in which instances the calcified bridge usually formed directly against the pulp-capping material. In a few cases there were dentin chips and necrotic debris, including degenerating red blood cells, between the bridge and Dycal. The first part of the bridge occasionally contained basophilic material suggestive of superficial necrosis. One hour after the Dycal base and catalyst are mixed the pH is 9.3; in 24 hours it reaches 11.7; and at 7 days the pH amounts to 12.1° We can speculate that the highly alkaline characteristics of the Dycal destroyed any microorganisms at the exposure site. Conceivably, this may account for the finding that Dycal-treated pulps fared better than the Durelon-treated ones. Another contributing factor may be the fact that Dycal stimulated a complete dentin bridge at the exposure site, although we do not consider it highly significant whether a bridge does or does not form. Of greater importance is the lack of inflammation and associated infection. In this study no direct correlation was observed between the size of the pulpal exposure and the amount of inflammation. Most of the exposures were 1 mm. in diameter, but there was a variation from slightly less than 1 to more than 2 mm. There was no detectable difference in the inflammatory outcome or in the number of dentin chips found within the pulps whether the exposure was carefully done with an explorer or made inadvertently with the bur. In spite of care, some dentin chips were found in practically all of the exposed pulps. The pulpal reactions of teeth with incompletely formed apices did not differ from those of teeth that were completely developed. Furthermore, the amounts of reparative dentin that formed appeared comparatively equal whether the pulp chamber was very large or very small. Whether a pulp was exposed before capping for 3 or for 51/, hours had no bearing on how it fared. Interpreting the presence of microorganisms in the sections stained by the Brown and Brenn method was difficult, and one should be careful to distinguish
100
McWalter,
El-Kafmwy,
and Mitchell
Oral Surg. July, 1973
bacteria from fragmented nuclei of neutrophils, hemosiderin granules, and other artefacts. Furthermore, viability of organisms could not be ascertained by this method. SUMMARY AND CONCLUSIONS
The pulps of seventy-four permanent teeth of four monkeys were exposed and left contaminated with saliva and plaque for 3 to 5$$ hours. The pulps then were capped either with Keflin (an antibiotic), Dycal (a calcium-hydroxide compound), or Durelon (a polycarboxylate cement). Varnish and then amalgam were inserted over these agents in order to assure an adequate seal. The teeth were surgically extracted at 15, 50, 100, and 200 days. Of twenty-five teeth treated with Keflin, only 8 per cent reacted satisfactorily at all study periods. Of the twenty-four teeth treated with Dycal, 96 per cent gave a satisfactory response. At the 50-day intervals and later, success in the Dycal-treated pulps was 100 per cent, with all teeth showing no inflammation and having complete dentin bridges as demonstrated by serial sections and Procion vital dye markings. The calcified bridge frequently formed directly against the Dycal. Durelon was used to cap twenty-five pulps. At the 15-day interval, success was limited, but at 50 days it rose to 75 per cent. Combining the loo-day and 200-day teeth, a 90 per cent successfigure was obtained for the Durelon-capped pulps. With this material a calcified bridge seldom formed at the exposure site, although reparative dentinogenesis did occur on adjacent dentinal walls and around dentin chips. This study substantiates the efficacy of Dycal as a pulp-capping agent. Although Durelon is not recommended for this purpose, its innocuous effect on the pulp is remarkable. Keflin proved to be too irritating to be of any value in vital-pulp therapy. REFERENCES
1. Stanley, H. R.: How Much Could We Learn? Justification for Establishing a Registry for Pulpal Pathology, ORAL Suao. 29: 265269, 1970. 2. Lawson, B. F., and Mitchell, D. F.: Pharmacologic Treatment of Painful Pulpitis, ORAL Suno. 17: 47-61, 1964. Treatment of Pulpitis: 3. Mullaney, T. P., Lawson? B. F., and Mitchell, D. F.: Pharmacologic A Continuing Investigation, ORAL &no. 21: 479-491, 1966. 4. Gardner, D. E., Mitchell, D. F., and McDonald, R. E.: Treatment of Pulps of Monkeys With Vancomycin and Calcium Hydroxide, J. Dent. Res. 50: 1273-1277, 1971. 5. Kakehashi, S., Stanley, H. R., and Fitzgerald, R. T.: The Effects of Surgical Exposures of Dental Pulps in Germ-free and Conventional Laboratory Rats, ORAL SURG. 20: 340-349, 1965. 6. Smith, D. C.: A New Dental Cement, Br. Dent. J. 125: 381-384, 1968. 7. Truelove, E. L., Mitchell, D. F., and Phillips, R. W.: Biologic Evaluation of a Carboxylate Cement, J. Dent. Res. 50: 166, 1971. Eli Lilly and Company: Personal communication, 1971. t * Glass, R. L., and Zander, H. A.: Pulp Healing, J. Dent. Res. 28: 97-107, 1949. 10: The L. D. Caulk Company, Milford, Delaware: Personal communication, April, 1972.
Reprint requeststo: Commander George M. MeWalter, 3628 Arcadian Court Castro Valley, Calif. 94546
DC, USN
UNIVERSITY
SCHOOL
OF DENTISTRY
In this histologic study, the pulps of seventy-four permanent teeth of four monkeys were mechanically exposed, contaminated with saliva, and capped. The teeth were extracted at 15, 50, 100, and 200 days. Of the teeth treated with Keflin (an antibiotic), only 8 per cent reacted satisfactorily. A calcium-hydroxide compound gave a 96 per cent satisfactory response. Combining only the lo&day and 200-day teeth, a polycarboxylate cement gave a 90 per cent success figure.
T
here is a widespread belief among dentists that, when pulpitis due to pulp exposure develops, the treatment indicated is ideally root canal therapy or, if this is not available, extraction. A problem with this attitude is that, for various reasons, root canal therapy is frequently not done. It is time consuming, it demands special skills, and, therefore, for many patients it becomes economically unfeasible. This is one reason for the estimated 250,000 or more teeth that are extracted per day in the United States.l Studies have shown that pulpitis may be reversible,2-* and, therefore, may be treated conservatively. If vital-pulp therapy fails, a choice can then be From a thesis submitted by the senior author to the faculty of Indiana University School of Dentistry in partial fulfillment of the requirements for the M.S.D. degree. The opinions and assertions contained herein are those of the writers and are not construed as official or as reflecting the views of the Navy Department or the naval servi large. *Assistant Professor, Department of Oral Diagnosis/Oral Medicine. **Professor and Chairman, Department of Oral Diagnosis/Oral Medicine.
90
Volumk; 36 Numbei 1
Pulp capping
in monkeys
91
Fig. 2. Photograph showing typical, mechanically exposed pulps.
made between exodontia and a root canal filling. The importance of this “vital” aspect of endodontic treatment cannot be overemphasized. Although innumerable pulp-capping agents have been advocated, the search for an ideal capping material has continued. The purpose of this investigation was to determine the reactions of contaminated pulps of permanent monkey teeth to direct pulp capping with a calcium-hydroxide compound, an antibiotic, and a polycarboxylate cement. Calcium hydroxide, usually in pure powder form mixed with some vehicle, has been the most popular of the pulp-capping agents. Since microorganisms seem to be a major factor in the eventual death of the p~lp,~ the rationale for including an antibiotic in the present study is obvious. Polycarboxylate cements have shown great promise in that they are claimed to have all the attributes of the two most popular cements in dentistry (zinc oxide and eugenol and zinc phosphate cement) without their disadvantages.6 A polycarboxylate cement was found to be almost nonirritating when implanted subcutaneously in rats or placed in deep cavities in monkey teeth.7 The use of this material as a pulp..I capping agent puts it to an extreme biologic test. METHODS AND MATERIALS Four young monkeys were used-a Macaca nemestrina, a Macaca speciosa, and two Macaca mulatta. The animals were tranquilized by intramuscular injection of 5 mg. of phencyclidine hydrochloride. * Anesthesia was then obtained by intravenous injection of 22 mg. of sodium pentobarbitalf per kilogram of body weight. Additional amounts were injected as needed. A full-mouth set of radiographs was made with the exposure settings of 60 KVP, 6 Ma., and 0.25 second. An increased vertical angulation was used to conform to the monkey’s jaw. Seventy-four pulp exposures were made in twenty-two anterior and fifty-two “Sernylan, Bio-ceutic Laboratories, Inc., St. Joseph, MO. tSomnopenty1, Pitman-Moore, Inc., Fort Washington, Pa.
Oral Surg. .July. 1973
posterior teeth. This included every pcrmancnt tooth present cxc*ept t,hc c~lisl)ids. A%class v preparation (&‘ig. 1) was made on the facial surfac? CJf ea(‘11 toot11 with an air-driven, No. 701, tapcretl fissure carbide bur at high spoctl untl~r a watc>r coolant. Short blasts of air lverc used to remove debris. A ~11 was made in the center of the cavity floor with a slowly rotating Xo. 4 round carbide bur until pink dentin indicated that an exposure was imminent. An explorer point was then used to make the exposure, entering the pulps approximately 1 mm. This careful procedure was used to minimize the number of dentin chips introduced into the pulp. An attempt was made to keep the diameter of the exposure about 1 mm. On a few occasions the exposure was inadvertently made with the bur. No aseptic procedures were used since contamination of the pulp was intentional. A water spray was used to clean dentin from the cavities, and saliva and materia alba, if present, were introduced into the exposure to contaminate the pulp. The pulps were left open to the oral environment for 3 to 51/, hours before the pulp capping was done. Then each cavity was cleansed with a spoon excavator and water spray. Usually, hemorrhage was induced by this procedure, and cotton pellets were used to control it. The three compounds used as the pulp-capping agents were a caIcium hydroxide compound (Dyeal”), a polycarboxylate cement (Durelonf ), and the antibiotic, sodium cephalothin (Keflint). Because each has a distinctive appearance, it was impossible not to know which agent was being used. Each agent was used in every third tooth available according to t,he l-32 numbering system for teeth. Both Dycal and Durelon were mixed on a 1:l base-liquid ratio. A layer of 0.5 mm. or more was left against the pulp. The Keflin powder was made into a paste with the use of sterile water. This was deposited on the pulp and a piece of gold foil covering all of the cavity floor was placed to lend support during the condensation of amalgam. After a layer of Copalite varnish was placed in the cavity covering the margins, an 8:5 mercury-alloy ratio of amalgam was inserted. The animals were maintained on a normal laboratory diet.5 Each monkey received at least one intraperitoneal injection of Procion Brilliant Red H-SBS,ll an in vivo hard-tissue marking agent. The dosage was 100 mg. per kilogram of body weight. The dye was used to determine the amount of reparative dentin deposited between the time of injection and the time of extraction. One monkey was injected 8 days after pulp capping, the second 30 days later, the third at 60 and 90 days, and the fourth 150 days after capping. The teeth were removed at intervals of 15, 50, 100, and 200 days after pulp capping. On the day of extraction, the oral cavity was examined and full-mouth
*L. D. Caulk Co., Milford, Del. tPremier Dental Products Co., Philadelphia, Pa. $Eli Lilly and Co., Indianapolis, Ind. $Monkey Chow, Ralston-Purina Co., St. Louis, MO. III. C. I., Organ&, Inc., Providence, R.I.
Volume 36 Number 1
Pulp capping
in monkeys
93
Fig. 8. Photomicrograph of a Keflin-treated pulp at 15 days. Notice the severe inflammation with abscess formation. (Hematoxylin and eosin stain. Original magnification, x35.)
radiographs were made. Alveolar bone on the facial surface was removed first with a mallet and chisel. Elevators and forceps were used to deliver the teeth carefully in order to avoid root fracture. Immediately after extraction, 3 mm. of the apical portions of the roots was’cut off with a bur rotated at high speed under a water spray. The specimens were then placed in 10 per cent formalin until ground down on one proximal surface to ensure adequate pulp fixation. Decalcification was accomplished with 5 per cent formic acid. Serial 7-pm thick paraffin sections were then cut through the extent of the cavity, including the exposure site. Alternate slides were stained with hematoxylin and eosin. Unstained sections were examined by fluorescent microscopy for Procion red markings. Selected sections were stained by the Brown and Brenn method to demonstrate the presence of bacteria in the pulps. Eight untreated teeth were also included in the study for control purposes. RESULTS Of the three materials, Durelon was the most easily delineated on the radiographs since it was distinctly radiopaque. Dycal was only slightly more radiopaque than the dentin. Keflin, since it was covered with gold foil, could not be distinguished under the alloys, Gross internal resorption was observed in two teeth capped with Keflin, one for 100 days and the other for 200 days. A radiolucency suggestive of external resorption appeared at the apex of another loo-day, Keflin-treated tooth. Otherwise, no significant roentgenographic abnormalities were noted. The microscopic findings are summarized in Table I. Pulp reactions to the capping agents were classified as satisfactory or unsatisfactory. A satisfactory response demonstrated complete absence of inflammation within the pulp or a scattering of a few inflammatory cells localized at the exposure site, with or without complete bridging. All pulps that demonstrated a moderate inflammatory response at the exposure site or severe inflammation with abscess
94
HcWalter,
Table
El-Knfmwy,
Oral Surg. July, 1973
md Mitchell
I. Summary of the microscopic findings
in seventy-four
permanent teeth capped
Kejlin
Tooth No.
Calcifio briaghg Inflammation Capped 15 days (Mg:;reM1, 21 teeth) 4 0” 8 Severe 12 Severe 0” 14 Severe 0 20 Severe 26 Severe t 31 Severe 0 Cappei 50 days (Mgt~feM6, 23 teeth) 4 10 14 2
Mild i Severe 0 Moderate 0 Severe Severe i 26 Severe Moderate ?I Cap&i 100 days (z$ey M5, 18 teeth) 7 Incomplete 10 Severe 15 Severe 0” 29 Severe 23 i 30 Severe 0 Cappei 200 days (M%;:;z M2, 12 teeth) 1’9”
Severe Severe Severe
28 *O = Absent. t- = Unsatisfactory. t+ = Satisfactory.
:: i
Pulp
response
Tooth No.
Inflammation
10 15 19 25 30
Moderate Severe Mild Moderate Severe Mild Moderate
-t
+t
f
5 8 12 15 20 24 28 31
Mild
0 0
Mild Moderate 0 Mild Moderate
2 8 13 18 24 26 1: 21 29
0 Mild Moderate Mild
formation or pulp necrosis were regarded as having responded unsatisfactorily to the capping agent. At 15 days, all of the seven Keflin-treated pulps demonstrated severe inflammatory reactions with abscesses (Fig. 2). At 50 days, only one of eight teeth similarly treated gave a satisfactory reaction. Six teeth were capped with Keflin for 100 days. Of these, only one gave a satisfactory response, two demonstrated severe inflammation with abscess formation, and three displayed complete necrosis. Multinucleated giant cells in association with internal resorption were observed in three of the Keflintreated teeth. At 200 days, all four of the Keflin-treated pulps demonstrated severe inflammation with abscesses. In one tooth, gross internal resorption had penetrated to the periodontal ligament. At 15 days, only two of seven pulps capped with Durelon responded satisfactorily. These showed a scattering of a few inflammatory cells at the exposure site. Three pulps showed inflammation of a moderate intensity, and the two other pulps showed severe inflammation with abscess formation. Of eight teeth capped with Durelon for 50 days, 6 responded satisfactorily. Of these, three demonstrated no inflammation, and three showed a few in-
Volume 36 Number L
with Keflin,
Durelon,
Pulp
capping
in monkeys
95
and Dycal
Dwebn
DYCQJ
Caloi$o bridgildg
Pulp response
Tooth No.
+
2 5 9 :i
Mild Mild Mild Mild Severe
+
29 24
Mild 0
0 0
Incomplete Incomplete Incomplete Incomplete 0 Incomplete i Incomplete Incomplete Incomplete Complete Complete Incomplete Incomplete Incomplete 0 Incomplete
2
Caloi$o
I I@ammatio*
bridging Incomplete . 0 0 Incomplete 8 0
i; 29
Complete Complete Complete Complete Complete Complete Complete
i 14 19 25 31
Complete Complete Complete Complete Complete Complete
i 13
4 ii’ 30
Pulp I response + + +
Complete Complete Complete Complete
flammatory cells at the exposure site. Incomplete bridging was evident in five instances. Usually, no true dentin bridge appeared to occur at the site of the exposure, but dentin walls adjacent to the exposure and dentin chips appeared to be the main nidus for bridging (Fig. 3). When no inflammation was present, the usual picture at the exposure site was a compressed zone of fibroblasts, collagen fibers, and some capillaries. At 100 days, all six Durelon-capped pulps showed bridging and no inflammation (Fig. 4). A few times when a bridge appeared to be complete, careful study of serial sections revealed some vital tissue penetrating through the calcified material. As in the 50-day specimens, dentin chips and adjacent pulpal walls seemed to be the main stimuli for dentinogenesis. Typically, between the Durelon and the bridge were necrotic debris, degenerated red blood cells, and occasionally dentin chips and vital tissue. At 200 days, one of the four Durelon-capped pulps had moderate inflammation. The integrity of the amalgam seal was suspect in this case since the amalgam fell out during extraction. Reaction in the three other pulps was considered to be satisfactory: two showed a few inflammatory cells at the exposure site, and the other pulp was free of inflammation. All three pulps showed incomplete bridging.
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Fig. 8. An example of no inflammation in a Durelon-treated pulp at 50 days. Some vital tissue is present on the cavity preparation side. Examination of the serial sections showed the dentin bridge to be incomplete. (Hemntoxylin and eosin stain. Original magnification, x35.) Fig. 4. Microscopic section of a Durelon-capped pulp at 100 days with no inflammation. Exceptionally, this bridge was complete. Some tissue debris is present on exposure side of the dentin bridge. (Hematoxylin and eosin stain. Original magnification, x35.)
Six of the seven teeth capped with Dycal for 15 days gave satisfactory responses. Two of them exhibited no inflammation, and four showed very mild inflammation. Usually, some Dycal was present either in the preparation area or in small amounts in the pulpal tissue within macrophages (Fig. 5). The Dycal was frequently observed in direct contact with vital tissue (Fig. 6). At other times, necrotic debris, including extravasated red blood cells, was seen under the pulp-capping material. One specimen capped with Dycal for 15 days showed severe inflammation with abscess formation. At 50, 100, and 200 days, all seventeen pulps capped with Dycal were free of inflammatory cells. Each had a complete dentin bridge (Figs. 7 and 8). The calcified material that first formed was usually amorphous with cellular inclusions; then irregular tubular dentin was present, and, finally, dentin of regular tubular structure appeared. Three sources of dentinogenesis were observed : from the dentinal walls near the exposure site; around dentin chips; and at the exposure site in contact with the Dycal (Fig. 7). Sections from twenty teeth were selected for a modified Brown and Brenn stain, All of the Keflin sections were negative for bacteria, even a pulp with complete necrosis. The only Dycal-treated pulp that demonstrated any bacteria was the one with severe inflammation at the 15-day interval. Gram-positive
Volume Number
36 1
Pulp capping in molzlceys 97
Fig. 5. Photomicrograph of a Dycal-capped pulp at 15 days with no intlammation. Many dentin chips are present. Vital pulp tissue is in direct contact with Dycal. (Hematoxylin and eosin stain. Original magnification, x35.) Fig. 6. Higher magnification of exposure site in Fig. 5 showing Dycal (Mao76 at the top) in direct contact with vital pulp tissue. (Hematoxylin and eosin stain. Original magnification, x250.)
bacteria were demonstrated in four of the Durelon-treated pulps, which also were inflamed to varying degrees. Gram-negative organisms were either absent or too difficult to identify. No microorganisms were observed in teeth without pulpal inflammation. The monkey that provided the E-day specimens was injected with Procion red 8 days after pulp capping. No specimens showed labeling. Dye was administered 30 days after capping to the animal that supplied the 50-day teeth. Good labeling of the Dycal-treated teeth was observed. The continuous fluorescent line in the calcified bridge indicated that a complete bridge was present at approximately 30 days after operation. The monkey supplying the KM-day teeth was injected with the dye at 60
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and Mitchell
Oral Surg. July, 1973
Fig. 7. An example of a complete dentin bridge and no inflammation in a Dycal-capped pulp at 100 days. Some Dyeal is present in the pulp. (Hematoxylin and eosin stain. Original magnification, x35.) Fig. 8. A typical Dycal-treated pulp at 200 days with no inflammation. Dentin bridge is complete and the Dycal (black) abuts directly against the calcified bridge. Notice the reparative dentinogenesis on the dentinal walls near the exposure, around dentin chips and at the exposure site in contact with the Dycal. (Hematoxylin and eosin stain. Original magnification, x35.)
and 90 days after capping. Where much tertiary dentin had formed, the labeling was usually good. Two distinct lines were usually seen. The amount of reparative dentin formed between 60 and 90 days seemed to be diminishing. Interestingly, two lines were seen in the dentin walls of canals of nontreated teeth. Procion red was administered to the animal providing the 200-day specimens at 150 days after operation. No marking was seen in the Keflin-capped teeth. The lines were vary faint, if seen at all, in the Dycal-treated and Durelon-treated teeth. This indicated that reparative dentin formation had ceased or markedly diminished in rate by 150 days after capping. DISCUSSION
All pulps used in this study were contaminated 3 to 51/s hours with saliva or plaque before pulp capping, since contact of microbial flora with the pulp is a major factor in pulpitis and pulp necrosis .5 We speculated that a broad-spectrum antibiotic, such as Keflin, would kill the microorganisms and thereby enhance the healing of the pulp. However, of the twenty-five teeth treated with this antibiotic, only two reacted satisfactorily. In selected sections stained to demonstrate bacteria, none was found, even in a tooth with complete pulpal necrosis. We can assume that, even though Keflin killed the bacteria, it was so severely irritating to the pulp that the tissue was irreversibly damaged. The pH of an aqueous solution of Keflin is 5.2.8
Volume 36 Number 1
Pulp
capping in monkeys
99
In the Durelon-treated teeth, only two of seven pulps had satisfactory reactions at the 15-day interval. The success rate rose to 75 per cent at 50 days. When the lOO-day and 200-day specimens were combined, a 90 per cent success figure was reached. The amalgam seal was suspect in the one tooth in the latter category where moderate inflammation was observed. In two instances a complete dentin bridge formed across the exposure site under Durelon ; however, these were exceptions. Frequently, an incomplete bridge formed, with vital tissue between it and the exposure site. Dentin chips and the adjacent pulpal walls apparently acted as niduses which stimulated dentinogenesis, and sometimes coalescence of this newly formed dentin resulted in calcified bridging. Some necrotic debris was occasionally observed at the exposure site. This was probably due to necrosis caused by the trauma of the mechanical exposure and not to irritation from the Durelon. Only one of the twenty-four teeth treated with Dycal gave an unsatisfactory response, and this was at the 15-day interval. At 50 days and later, none of these teeth exhibited any inflammation and all demonstrated complete dentin bridges, confirmed by the study of serial sections and vital-dye markings. The pulpal reaction to Ca(OH), pastes typically leaves a necrotic zone between the bridge and the paste.9 This often was not the case with Dycal, at the intervals studied, in which instances the calcified bridge usually formed directly against the pulp-capping material. In a few cases there were dentin chips and necrotic debris, including degenerating red blood cells, between the bridge and Dycal. The first part of the bridge occasionally contained basophilic material suggestive of superficial necrosis. One hour after the Dycal base and catalyst are mixed the pH is 9.3; in 24 hours it reaches 11.7; and at 7 days the pH amounts to 12.1° We can speculate that the highly alkaline characteristics of the Dycal destroyed any microorganisms at the exposure site. Conceivably, this may account for the finding that Dycal-treated pulps fared better than the Durelon-treated ones. Another contributing factor may be the fact that Dycal stimulated a complete dentin bridge at the exposure site, although we do not consider it highly significant whether a bridge does or does not form. Of greater importance is the lack of inflammation and associated infection. In this study no direct correlation was observed between the size of the pulpal exposure and the amount of inflammation. Most of the exposures were 1 mm. in diameter, but there was a variation from slightly less than 1 to more than 2 mm. There was no detectable difference in the inflammatory outcome or in the number of dentin chips found within the pulps whether the exposure was carefully done with an explorer or made inadvertently with the bur. In spite of care, some dentin chips were found in practically all of the exposed pulps. The pulpal reactions of teeth with incompletely formed apices did not differ from those of teeth that were completely developed. Furthermore, the amounts of reparative dentin that formed appeared comparatively equal whether the pulp chamber was very large or very small. Whether a pulp was exposed before capping for 3 or for 51/, hours had no bearing on how it fared. Interpreting the presence of microorganisms in the sections stained by the Brown and Brenn method was difficult, and one should be careful to distinguish
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Oral Surg. July, 1973
bacteria from fragmented nuclei of neutrophils, hemosiderin granules, and other artefacts. Furthermore, viability of organisms could not be ascertained by this method. SUMMARY AND CONCLUSIONS
The pulps of seventy-four permanent teeth of four monkeys were exposed and left contaminated with saliva and plaque for 3 to 5$$ hours. The pulps then were capped either with Keflin (an antibiotic), Dycal (a calcium-hydroxide compound), or Durelon (a polycarboxylate cement). Varnish and then amalgam were inserted over these agents in order to assure an adequate seal. The teeth were surgically extracted at 15, 50, 100, and 200 days. Of twenty-five teeth treated with Keflin, only 8 per cent reacted satisfactorily at all study periods. Of the twenty-four teeth treated with Dycal, 96 per cent gave a satisfactory response. At the 50-day intervals and later, success in the Dycal-treated pulps was 100 per cent, with all teeth showing no inflammation and having complete dentin bridges as demonstrated by serial sections and Procion vital dye markings. The calcified bridge frequently formed directly against the Dycal. Durelon was used to cap twenty-five pulps. At the 15-day interval, success was limited, but at 50 days it rose to 75 per cent. Combining the loo-day and 200-day teeth, a 90 per cent successfigure was obtained for the Durelon-capped pulps. With this material a calcified bridge seldom formed at the exposure site, although reparative dentinogenesis did occur on adjacent dentinal walls and around dentin chips. This study substantiates the efficacy of Dycal as a pulp-capping agent. Although Durelon is not recommended for this purpose, its innocuous effect on the pulp is remarkable. Keflin proved to be too irritating to be of any value in vital-pulp therapy. REFERENCES
1. Stanley, H. R.: How Much Could We Learn? Justification for Establishing a Registry for Pulpal Pathology, ORAL Suao. 29: 265269, 1970. 2. Lawson, B. F., and Mitchell, D. F.: Pharmacologic Treatment of Painful Pulpitis, ORAL Suno. 17: 47-61, 1964. Treatment of Pulpitis: 3. Mullaney, T. P., Lawson? B. F., and Mitchell, D. F.: Pharmacologic A Continuing Investigation, ORAL &no. 21: 479-491, 1966. 4. Gardner, D. E., Mitchell, D. F., and McDonald, R. E.: Treatment of Pulps of Monkeys With Vancomycin and Calcium Hydroxide, J. Dent. Res. 50: 1273-1277, 1971. 5. Kakehashi, S., Stanley, H. R., and Fitzgerald, R. T.: The Effects of Surgical Exposures of Dental Pulps in Germ-free and Conventional Laboratory Rats, ORAL SURG. 20: 340-349, 1965. 6. Smith, D. C.: A New Dental Cement, Br. Dent. J. 125: 381-384, 1968. 7. Truelove, E. L., Mitchell, D. F., and Phillips, R. W.: Biologic Evaluation of a Carboxylate Cement, J. Dent. Res. 50: 166, 1971. Eli Lilly and Company: Personal communication, 1971. t * Glass, R. L., and Zander, H. A.: Pulp Healing, J. Dent. Res. 28: 97-107, 1949. 10: The L. D. Caulk Company, Milford, Delaware: Personal communication, April, 1972.
Reprint requeststo: Commander George M. MeWalter, 3628 Arcadian Court Castro Valley, Calif. 94546
DC, USN