Bacterial contamination as a factor influencing the toxicity of materials to the exposed dental pulp

endodontics

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Bacterial contamination as a factor influencing the toxicity of materials to the exposed dental pulp Andrew Watts, Ph.D., B.D.S., L.D.S.R.C.S., and Robert C. Paterson, M.D.S., Ph.D., Glasgow, Scotland

DEPARTMENT OF CONSERVATIVE DENTISTRY, GLASGOW DENTAL HOSPITAL AND SCHOOL

Three materials, which had been previously found to be toxic when applied as pulp-capping agents in

conventional rat molar pulps, were retested in germ-free rats. All produced much more favorable responses

in the pulp, with a lack of inflammation and the presence of dentine bridges in the majority of teeth. It

appears that much of the pulp damage previously attributed to the chemical toxicity of materials may be

caused by the presence of bacteria. The design of studies intended to evaluate the response of the pulp to

materials should include staining techniques that will detect the presence of bacteria on the floors of test

cavities.

(ORAL SURG ORAL MED ORAL PATHOL 1987;64:466-74)

I n previous reports’* * a zinc polycarboxylate cement, aluminum hydroxide powder, and titanium dioxide powder have been shown to be irritant to the rat molar pulp. Pulp capping with these materials resulted in necrosis and apical inflammation, with very few dentine bridges being formed.

These studies were carried out in conventional rats with a complete oral flora. When sections were stained to demonstrate the presence and distribution of bacteria, numbers of organisms were found to be present at the margins of the amalgam restorations, placed over the experimental pulp-capping materials, and also on the cavity base and at the pulp wound surface.

Experiments with germ-free rats have shown that zinc phosphate cement produced good results when used to cap mechanically exposed pulps, with a high incidence of dentine bridge formation. Even when

Part of the material contained in this paper was included in the Ph.D. theses submitted to the University of London by Dr. Watts and Professor Paterson.

466

silicate cement was used, there was evidence of repair in the form of bridges, although these were formed at some distance from the original exposure site, from which they were separated by a zone of necrotic pulp tissue. 3 Similar studies with pulp- capping cements containing corticosteroids and anti- biotics have shown poor results in the conventional animals and, in contrast, good responses in germ-free animals.4

The objective of the present study was to deter- mine the response of the mechanically exposed germ- free rat molar pulp to the same zinc polycarboxylate cement that was used in our conventional animal studies and also to aluminum hydroxide and titanium dioxide.

METHODS

The same Fiberglas germ-free breeding/surgical isolators as described previously5 were used for this study. Albino germ-free rats of the Fischer strain were imported as breeding trios (Charles River Breeding Laboratories, Wilmington Massachusetts). Experimental animals were bred within the germ-

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Bacterial contamination and exposed dental pulp 467

Table I. Inflammation/necrosis scores

infiammation Necrosis

No. of Surface Coronal pulp Root canal Periapical tissue Coronal pulp Root canal

Material teeth cells + ++ + ++ + -+I- + ++ + ++

Poly c Conv * (28 days) Al (OH)3 Conv (28 days) Ti 02 Conv (28 days) Poly c Germ-free (7 days) (28 days) Al (OH) 3 Germ-free (1 day) (2 days) (7 days) (28 days) Ti 02 Germ-free (1 day) (2 days) (7 days) (28 days)

18 -

4

4

8 5 12 2

8 8 8

12

-

2 1

1

1 -

- 2 -

- -

-

1 4

4

4 3

6 6 6

12

6 6

- 5 6

+=Gradel. ++ = Grade 2. *Paterson’ 1976 data restored.

free isolator. The rats were large enough for surgery when they were 8 weeks old. They were anesthetized in the same isolator with fluothane and oxygen. The pulps of the maxillary first and second molar teeth were exposed. Cavities were prepared with a No. 33% inverted cone bur (IS0 006) revolving at low speed (not more than 1,500 rpm) and used intermit- tently. Pulp exposure was achieved by the application of firm pressure with a sharp right-angled probe on the cavity floor. The bleeding exposure was irrigated and dried with paper points. The zinc polycarboxylate cement (Poly C, De Trey Dentsply, Weybridge, Surrey, England) was applied to the exposure and allowed to set. Any excess was trimmed from the cavity walls, and the cavity was sealed with silver amalgam. The aluminum hydroxide and the titanium dioxide were mixed to a stiff paste with distilled water and applied to the pulp exposure. A Dycal (L.D. Caulk Co., Milford, Delaware) lining was applied and allowed to set. The cavity was sealed with silver amalgam. The animals were allowed to recover and were maintained in the germ-free isolator for the remainder of the experiment. They were killed in groups after 1,2,7, and 28 days.

The maxillas were dissected out and split through the midline palatal suture. They were decalcified and processed for wax embedding. Sections were cut in a mesiodistal plane through the first and second molars at a thickness of 8 microns. They were stained with hematoxylin and eosin. Examination was carried out at low and high magnification. Inflamma- tion and necrosis in the coronal pulp, root canals, and periapical tissues were recorded. A note was made of the presence of calcification as a bridge at the exposure site or as calcification in the coronal pulp and root canals. All criteria except bridge formation were graded as follows:

Znjlammation/necrosis Surface cells:

Inflammatory cells lying at the wound surface (i.e., not infiltrating pulp tissue) were recorded as surface cells.

Coronal pulp, root canals: Grade l-Localized (i.e., less than one half

involved). Grade 2-Extensive (i.e., more than one half

involved).

468 Watts and Paterson Oral Surg. October 1987

Bacteriologic nionitoring of the germ-free isolators

Mixed samples consisting of fresh feces mixed with drinking water were removed from the isolator at weekly intervals for monitoring. These were inoculated into a range of media and subjected to both aerobic and anaerobic conditions of growth at body and room temperature, for at least 14 days.

At the conclusion of each experiment one of the sacrificed animals, chosen at random, was dissected within the isolator. Samples of all its major organs were removed, inoculated into media, and subjected to the same conditions of growth as described for the fecal samples.

RESULTS

The histologic appearances are illustrated in Figs. 1 to 8, and the numerical results are presented in Tables I and II. The data from our .previous reports on the same materials in conventional animals have been included for comparison.

Poly c

Seven days after pulp exposure. Eight teeth were available for examination. Early necrosis of the tissue surface of the pulp wound was visible in four teeth. Inflammatory cells, mainly polymorphonuclear leukocytes, were present in the surface debris overlying the exposure in five teeth (Fig. 1).

Twenty-eight days aftkr pulp exposure. Twelve teeth were available for examination. In seven teeth dentine bridges Were present at the exposure site (Fig. 2). Of the remaining teeth, necrosis of pulp tissue extending into the root canal was observed in two specimens. In both cases the amalgam fillings had been lost (Fig. 3). Inflammatory cells (a mixture of polymorphonuclear leukocytes and small round cells) were observed at the original exposure sites in two teeth.

Aluminum hydroxide

One day after pulp exposure. Eight teeth were available for examination. Limited necrosis of the surface tissue of the pulp was observed in six teeth (Fig. 4).

Two days after pulp exposure. Eight teeth were available for examination. Early necrosis of the superficial cells in the pulp was observed in five teeth. In ane of the remaining teeth, filling material had been forced into the root canal, resulting in some necrosis of the pulp tissue.

Seven days after pulp exposure. Eight teeth were

Fig. 1. High magnification of pulp exposure in germ- free rat molar that had been pulp capped with Poly C 7 days previously. A mass of dentine fragments and cement particles is surrounded by inflammatory cells, which are mainly polymorphonuclear leukocytes. (Magnification, X450.)

Periapical tissues: Grade l-One root involved. Grade ~-TWO or more roots involved.

(Only infiltration of the pulp tissues by inflammatory cells ranked as positive inflammation scores. Hyperemia or inflammatory cells contained in blood vessels were not scored as positive.) Calcification A bridge was scored only if observed in more than 75% of the sections through the exposure site. Coronal pulp, root canal calcification:

Grade l-Localized (i.e., less than one half involved).

Grade 2-Extensive (i.e., more than one half involved).

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Fig. 2. First molar from germ-free rat that had been pulp capped with Poly C 28 days previously. There is some necrotic tissue adjacent to the exposure site. Well-formed dentine bridges are present. (Magnification, x60.)

Table II. Dentine bridge formation/calcification

Calcification

Poly c

Conv *

(28 days) Al (OH)3 Cow

(28 days) Ti02

Conv

(28 days) Poly c Germ-free

(7 days) (28 days)

Al (OH)3 Germ-free

(1 day) (2 days) (7 days) (28 days)

Ti 02 Germ-free

(1 day)

(2 days) (7 days) (28 days)

No. of teeth Dentine bridge

18 6

4 -

4 I

8 -

12 7

8 -

8 -

8 2

12 I2

8 -

I -

8 I

7 7

Coronal pulp Roar canal

+ ++ + ++

- - - -

4 4 -

3 - 3

-

2 I 3

- - - - -

7 - - - - - -

- - - - -

8 - 1 - - - -

+ =Grade I. ++ = Grade 2. *Paterson1 1976 data restored.

470 Watts and Paterson Oral Surg. October 1987

Titanium dioxide

One day after pulp exposure. Eight teeth were available for examination. Minimal superficial necrosis was present in the majority of specimens.

Two days after pulp exposure. Seven teeth were available for examination. In the majority of pulps a minimal superficial necrosis was observed. Polymor- phonuclear leukocytes were observed in the surface debris in one specimen. Capping material within phagocytic cells was observed in two cases (Fig. 7).

Seven days after pulp exposure. Eight teeth were available for examination. Necrosis of the coronal pulp tissue was observed in the majority of specimens. An early dentine bridge was present in one tooth. In all of the remaining specimens irregular calcific material was present in the coronal pulps. In more than half of the specimens capping material was observed within phagocytic cells.

Twenty-eight days after pulp exposure. Seven teeth were available for examination. Minimal superficial pulp necrosis with calcific bridge formation on the pulpal aspect was noted in the majority of specimens (Fig. 8). Where deeper necrosis was present, this was limited to the root canal beneath the original exposure site. The remaining pulp had calcific barriers. Particles of the capping material were observed in macrophages in the pulps of two teeth.

DISCUSSION

The germ-free isolator techniques used in the present study have been employed over a number of years, with some success. The only difficulties that have been encountered have been as a result of the gamma irradiation used to sterilize instruments and materials. This can alter the properties of plastic items, rendering them brittle. The effects on dental materials are normally minimal, but in the current study the zinc polycarboxylate cement used (Poly C) was supplied in a pack containing a lining and a luting liquid. These were intended to allow the preparation of cements of different viscosities for the different clinical applications. The lining liquid was a much more viscous fluid. After irradiation, it had set hard, presumably as a result of a polymerization reaction. The luting liquid was not affected and was used in this study.

The importance of bacterial presence in the response of the pulp to mechanical trauma is now well recognized. The classic early study by Kake- hashi et al6 showed that in germ-free animals even large exposures left open to the oral cavity healed normally. This observation was confirmed by Pater- son4 again using germ-free rats.

Fig. 3. Second molar from germ-free rat that had been pulp capped with Poly C 28 days previously. The amalgam filling was lost. The pulp chamber contains debris. The pulp tissue is completely necrotic. (Magnification, X60.)

available for examination. Superficial minimal pulp tissue necrosis was noted in the majority of teeth. There was evidence of early calcific repair in all of the teeth. In two cases complete early dentine bridges were present. In one specimen scattered polymorphonuclear leukocytes were observed at the original exposure site. In two cases phagocytic cells containing capping material were also observed lying super- ficially.

Twenty-eight days after pulp exposure. Twelve teeth were available for examination. In each of the teeth a zone of superficial pulp necrosis was noted, with calcific bridges on the pulpal aspect. In all but one of these bridges, regular reparative dentine was present on the pulpal aspect (Fig. 5). In one case particles of capping material were observed within remnants of phagocytic cells lying in the superficial necrotic tissue (Fig. 6).

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Fig. 4. Pulp exposure in a germ-free rat that had been pulp capped with aluminum hydroxide 1 day previously. There is minimal necrosis in the superficial layers of the pulp. (Magnification, X190.)

Fig. 5. First molar from a germ-free rat that had been pulp capped with aluminum hydroxide 28 days previously. There are well-formed dentine bridges present. The pulp appears normal. (Magnification, X60.)

The early germ-free experiments were extended by Kakehashi et al.,’ who studied the effect of prednis- olone on the exposed germ-free pulp and observed that it made little difference in the healing observed previously. Paterson4 applied Dycal (L. D. Caulk) to both conventional and germ-free rat molar pulps. Although Dycal allowed dentine bridge formation in a high percentage of teeth in conventional rats, inflammatory cells were observed in a number of pulps. This was not the case in germ-free animals, where perfect bridge formation had occurred. Simi- lar work with a corticosteroid cement and silicate and phosphate cements have been referred to at the beginning of this article.

These studies strongly supported the view that bacteria may modify the response of the exposed pulp to dental materials.

Brannstrom and Nyborg8 reported that they had restained the material from an earlier study in which they had reported an adverse response to zinc phosphate cement. They observed a bacterial presence on the cavity walls and floors. These bacteria could have remained from the original carious lesion, become smeared around the cavity walls during preparation, or gained access during the life of the test restoration as a result of microleakage at the margins. Brann- Strom and Nyborg concluded that much of the early work on pulp response to materials, in which no

472 Watts and Paterson Oral Slug. October 1987

Fig. 6. Higher magnification of the exposure site from specimen shown in Fig. 5. There is a small zone of necrosis superficial to the dentine bridges. Particles of capping material can be seen within the remnants of phagocytic cells (arrowsj. Calcific material surrounds dentine fragments. (Magnification, X800.)

Fig. 7. Pulp exposure from a germ-free rat treated 2 days previously with titanium dioxide. The material appears as a dark mass. There is necrosis of superficial tissue and scattered inflammatory cells (scored as “surface cells”). These are mainly polymorphonuclear leukocytes. Some of these appear to have ingested particles of the capping material. (Magnification, X500.)

bacterial staining had been carried out, should be interpreted with caution. Similar findings were reported by us when we restained material from our earlier studies on the toxicity of materials.9 In the majority of teeth in which an unfavorable pulp response had been noted, bacteria were present on the floor of the cavity near the original exposure site.

Two of the materials tested in the present study (aluminum hydroxide and titanium dioxide) had been screened out in a pilot studylo as too toxic for use in pulp-capping cements. These results were

surprising in that many authors attribute the favorable responses seen to calcium hydroxide to the presence of the hydroxyl ion. Further, titanium dioxide is one of the components of Dycal (L. D. Caulk). In the absence of bacteria, considerably more favorable responses were seen, with calcific repair in evidence by the seventh day and no evidence of pulpal inflammation. The responses with the zinc polycarboxylate cement were better in the germ-free animals than in the earlier conventional animal study. However, the results in the germ-free animals were not as favorable as those seen with the other

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Fig. 8. First molar from germ-free rat that had been pulp capped with titanium dioxide 28 days previously. There is minimal necrosis of the pulp tissue in contact with the capping material. well-formed dentine bridges are present. The remaining pulp appears normal. (Magnification, X60.)

materials. There were several necrotic pulps, and not all teeth had formed dentine bridges. In some of these specimens there was evidence of diet in the pulp chamber, suggesting that the restorations may have leaked and allowed food packing. We have observed previously*’ that this can produce necrosis of pulps in the germ-free rat. It is perhaps worthy of note that we did find some necrosis and reduction of calcific repair when a glass ionomer cement was tested in germ-free rats in a similar study.** This material is also prepared with polyacrylic acid. It is therefore possible that some limited chemical toxicity to the pulp may be associated with cements that have a polyacrylic acid base.

This study completes a series of experiments in which different materials have been applied to the exposed pulps of germ-free rats. In the earlier studies proprietary materials intended for use as direct or indirect pulp caps were used (i.e., calcium hydroxide and corticosteroid-antibiotic cements). Next, materials that were considered to be irritant to the pulp were tested (i.e., silicate and phosphate cement). In this final study, materials found toxic to the exposed pulp were examined. With all of these mateiials, better results were achieved in the germ- free animals. In the absence of bacteria, any damage produced by materials seems to be limited to the superficial parts of the pulp. The most common finding is of superficial necrosis of tissue without inflammatory infiltration of the adjacent vital pulp.

In reexamining our conventional rat material, as mentioned above, we have observed the presence of bacteria at or near the exposure site in the majority of cases in which there was an adverse response to a

material. It is important to remember that bacteria, particularly the gram-negative organisms, are diffi- cult to stain in histologic sections. StanleyI has suggested that for every organism detected in this way, there are at least 25,000 undetected. Staining techniques detect only a small fraction of the bacteria that are actually present. It appears reasonable to postulate that chemical damage produced by materials is limited in extent; where extensive damage and inflammatory infiltration of pulp tissue are observed, bacterial contamination must be sus- pected.

It would therefore appear essential in the testing of pulp responses to materials to include techniques designed to detect the presence of bacteria. Watts* has discussed the difficulties involved in the staining of bacteria in decalcified specimens. Much work is required before these techniques can be considered reliable. In the meantime, the presence of bacterial contaminants must be ruled out before a material can be considered toxic to the pulp.

In our view, it could be argued that the “toxicity” of most dental materials to the pulp may be more of a reflection of their sealing ability than their chemical properties, such as acidity.

CONCLUSIONS

1. The responses of the rat molar pulp to mechanical exposure and pulp capping in germ-free animals were more favorabie than previously observed in conventional animals. Bacteria were therefore considered responsible for much of the damage previously attributed to chemical toxicity.

2. All studies designed to assess the response of

474 Watts and Paterson

the pulp to dental materials should include a regimen designed to detect the presence of bacteria on the cavity floor.

The animal work for this study was carried out at Guy’s Hospital Medical School, London. Technical support was funded through a project grant from the Medical Research Council. We are grateful to Mr. S.K. Pountney for his skilled maintenance of the isolators and the processing of the tissues for histologic study. Prof. A.H.R. Rowe provided enthusiastic support in the early stages of the program of which this study forms a part. Mr. R.F. Wilson, Dental Clinical Research Laboratories, Guy’s Hospital, gave much valued advice. The Oral Biology Unit, Glasgow Dental Hospital and School, provided laboratory accommodation. Mr. J. Gillespie and Mr. R. Foye prepared the photomicrographs.

REFERENCES

1. Paterson RC. The reaction of the rat molar pulp to various materials. Br Dent J 1976;140:93-6.

2. Watts A. Factors influencing the response of the artificially exposed dental pulp to different materials in rat and dogs [Ph.D thesis]. London: University of London, 1981.

3. Watts A. Bacterial contamination and the toxicity of silicate and zinc phosphate cements. Br Dent J 1979;146:7-13.

4. Paterson RC. Bacterial contamination and the exposed pulp. Br Dent J 1976;140:231-6.

5. Paterson RC, Jones RW, Cook R, Garriock D. A simplified Fiberglas germ-free isolator with facilities for performing dental surgery on rats. Lab Anim 1970;4:233-40.

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Oral Surg. October 1987

Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. ORAL SURG OVAL MED ORAL PATHOL 1965; 20:340-9. Kakehashi S, Stanley HR, Fitzgerald RJ. The exposed germ-free pulp: effects of topical corticosteroid medication and restoration. ORAL SURG ORAL MED ORAL PATHOL 1969; 27:60-7. Brannstrom M, Nyborg H. Bacterial growth and pulpal changes under inlays cemented with zinc phosphate cement and Epoxylite CBA 9080. J Prosthet Dent 1974;31:556-65. Watts A, Paterson RC. Bacterial contamination and the “toxicity” of materials to the exposed pulp. ORAL SURG ORAL MED ORAL PATHOL 1983;56:542-8. Watts A, Paterson RC. Simple metallic compounds as pulp capping agents. ORAL SURG ORAL MED ORAL PATHOL 1977; 44:285-92. Paterson RC. An investigation into the histological changes produced in the rat molar pulp in response to caries and various microorganisms [M.D.S. thesis]. London: University of London, 1978. Paterson RC, Watts A. Toxicity to the pulp of a glass ionomer cement. Br Dent J 1987;162:110-2. Stanley HR. Importance of the leucocyte to dental and pulpal health. J Endod 1977;3:334-41.

Reprint requests to:

Dr. Andrew Watts Department of Conservative Dentistry Glasgow Dental Hospital 378, Sauchiehall St. Glasgow G2 352, Scotland

Bacterial contamination as a factor influencing the toxicity of materials to the exposed dental pulp

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