0099-2399/89/1512-0591/$02.00/0 JOURNAL OF ENDODONTICS Copyright �9 1989 by The American Association of Endodontists

Printed in U.S.A. VOL. 15, NO. 12, DECEMBER 1989

Experimental Bacterial Anachoresis in Dog Dental Pulps Capped with Calcium Hydroxide

Dimitrios Tziafas, DDS, DrD

The pulps of 36 permanent dog teeth were mechan- ically exposed and capped with Dycal, calcium hy- droxide powder mixed with saline, or Teflon. At 2, 14, and 28 days postoperatively, nine teeth treated with the materials were extracted (treated control teeth): A suspension of streptococci was then in- jected intravenously. Twenty-four h later the dogs were killed and both the 27 treated teeth (experi- mental group) and 6 unoperated control teeth were removed in tissue blocks. Tissue sections were ex- amined for the presence of bacteria, hard tissue formation, inflammatory cell response and necrosis. Bacteria were not observed in the unoperated and treated control teeth or in three of four teeth capped with Teflon for 29 days. In all the remaining speci- mens colonies of gram-positive cocci were found.

The localization and fixation of blood-borne microorganisms (or other materials) in zones of inflammation, a phenomenon named anachoresis, was first described in 1867. For pulpal inflammation, anachoresis has been experimentally investi- gated in order to explain the presence of microorganisms in unexposed pulps (1-4). It has been demonstrated that pulpal inflammatory reactions caused by dental procedures may attract microorganisms to pulp sites via the hematogenous route (5-10).

Successful pulp-capping treatment with calcium hydroxide- containing materials is characterized by the formation of a hard tissue barrier bridging the exposure site and overlaying a healthy pulp (11). In many cases the treated pulp is necro- tized with or without bridge formation. Failure of pulp- capping treatment may be explained by several factors: (a) age and inflammatory status of the pulp (12-14), (b) bleeding of the tissue at the time of placing the capping material (15), (c) severe chemical injury of the pulp due to the intense alkalinity of calcium hydroxide (16, 17), and (d) infection of pulp by oral microflora (18-21). The role played by blood- borne bacteria in the prognosis of pulp-capping treatment with calcium hydroxide base materials is unknown.

The purpose of this experimental study was to investigate whether the calcium hydroxide treated pulp could be infected by blood-borne microorganisms during the initial postopera- tive period.

591

MATERIALS AND METHODS

Three healthy dogs weighing 13 to 16 kg and 8 to 12 months of age were used. The animals were anesthetized with pentothal and intubated with a cuffed endotracheal tube at the beginning of the experimental procedure. Four types of teeth; second upper incisors, third upper and lower incisors, upper and lower canines and first upper and lower molars were used, as listed in Table 1. The teeth were divided into three groups: unoperated control, treated control, and exper- imental group. The teeth, which were intact and with healthy periodontium, were isolated with cotton rolls, polished, and washed with 0.5 % chlorhexidine.

Experimental Procedure

In the third incisors, canines, and first molars of both treated control and experimental groups, buccal class V cav- ities were prepared by means of a #31 inverted cone carbide bur in an air turbine handpiece with water spray. The pulps were exposed with a ~00 round bur, which was operated at low speed, producing a wound the size of the cutting edge of the bur. The cavities were washed with sterile saline and dried with sterile cotton pellets. Light pressure with cotton pellets was applied to control hemorrhaging.

The exposed pulps were capped with Dycal (L. D. Caulk Co.), calcium hydroxide powder mixed with sterile saline (Merck), or sterile disks of Teflon. All teeth were restored with zinc oxide-eugenol cement and amalgam. The teeth of the experimental and the treated control groups were treated sequentially so that three observation periods distributed among the three dogs were available. At 2, 14, and 28 days postoperatively, the nine third incisors of the treated control group were extracted and fixed in 10% neutral buffered formalin. Then 4 ml of a milky suspension of a-streptococci obtained from a positive human endodontic culture were injected into a hindleg vein. The animals were killed by an overdose of pentothal 24 h after the injection of bacteria.

The jaws were immediately dissected and the apical part of the roots of the experimental and the unoperated control teeth were removed. The tissues were fixed in 10% neutral buffered formalin. Intact restorations were observed in the treated teeth at the time of fixation. The tissues were decalcified in 5% trichloroacetic acid and processed for histological sectioning. Eight nonserial sections 6-gm thick were cut, labiolingually, through the exposure site. Three sections were stained with

592 Tziafas

Mayers' hematoxylin and eosin and five sections with a modified gram stain according to the method of Brown and Brenn.

I listological Assessment

The following criteria were used to assess the specimens: 1. l lard tissue formation. The presence of a calcified bar-

rier (continuous or discontinuous) around the exposure site in all examined sections was characterized as calcified bridge formation.

2. Inflammatory cell response. Inflammatory cell infiltra- tion of the pulp tissue was classified as: none, absence of inflammatory cells; slight, a few scattered inflammatory cells; moderate, moderate inflammatory cell infiltration around the exposure site; or severe, heavy inflammatory cell infiltration of the coronal pulp, abscess formation.

3. Necrosis. Complete tissue degeneration of half or more of the coronal pulp was characterized as necrosis.

4. Bacterial infiltration. The microbiological condition of the pulp was classified as follows:

Marked Bacterial Infiltration

Journal of Endodontics

No Bacterial Infiltration

Total absence of bacterial staining reaction in the extravas- cular space of the pulp was demonstrated.

RESULTS

Gram-positive or gram-negative bacteria were not identi- fied along the cavity wails or within the cut dentinal tubules in any operated teeth.

Unoperated Control Teeth

Microorganisms were not detected in the histologically normal pulps of the six intact second incisors; bacteria were only seen within pulpal blood vessels.

Treated Control Teeth

Bacterial staining reactions were not identified in the pulp of the treated third incisors which had been extracted before the induction of bacteremia.

Marked bacterial infiltration around the exposure site was observed; a few scattered bacteria in the underlying pulp. Experimental Teeth

Slight Bacterial Infiltration The results are given in Table 2.

A few scattered bacterial colonies around the exposure site were evident; no or a few scattered bacteria in the underlying pulp.

TABLE 1. The types of teeth used in each of the three groups

Type of Teeth No. of Teeth Group

Second incisors 6 Unoperated control Third incisors 9 Treated control Third incisors 3 Experimental Canines 12 Experimental First molars 12 Experimental

Pulpal Reactions

A number of teeth that had been treated with Dycal or calcium hydroxide-saline paste showed a reaction zone close to the wound surface, with no evidence of calcified bridging. The underlying pulp was normal except for occasional scat- tered inflammatory cells and congested blood vessels (Figs. 1 and 2). The reaction zone was of variable width and composed of capping materials, remnants of blood, dentin fragments, areas of tissue coagulation, traces of postoperatively formed hard tissue, inflammatory cells, area of hemorrhage, and edema. Inflammatory cells were predominantly neutophils: a

TABLE 2. The microbiological condition of the pulp of the experimental teeth in relation to their histopathological status

Capping Postoperative Bacterial infiltration Interval

Material (days) Marked Slight No Pulp Response

Dycal

3 3 15 2

29 2

3 3 Ca(OH)2-saline 15 3

29 1

3 3 15 2

Teflon 29

Moderate inflammation Moderate inflammation Slight inflammation with HTF* 1 moderate inflammation, 1 severe inflammation with HTF Slight inflammation with HTF

2 moderate inflammation, 1 severe inflammation 1 moderate inflammation, 2 slight inflammation with HTF Necrosis 1 slight inflammation with HTF, 1 moderate inflammation

with HTF

Moderate inflammation Moderate inflammation Slight inflammation No inflammation

* HTF, hard t issue formation.

Vol. 15, No. 12, December 1989 Anachoresis in Capped Pulps 593

FIG 2. Pulp exposure treated with Dycal for 29 days. Moderate inflammatory cell response accompanied by soft tissue disturbances beneath the exposure site (hematoxylin-eosin; original magnification x 16).

F~G 1, Pulp exposure treated with Dycal for 15 days. A, A thin zone of coagulation necrosis overlaying a hyperemic pulp with slight inflam- matory infiltration; traces of postoperatively formed hard tissue (he- matoxylin-eosin; original magnification x 16). B and C, Colonies of bacteria localized respectively in areas 1 and 2 outlined in A (Brown and Brenn; original magnification x 400).

few lymphocytes and macrophages were also seen. Hard tissue barrier formation, completely bridging the exposure site, was found in four specimens (Figs. 3 and 4). In these cases the calcified tissue was in direct contact with normal pulp which, however, showed increased vascularity and scattered inflam- matory cells.

The teeth which had been capped with Teflon showed a very limited reaction zone which was composed of dentin fragments, inflammatory cells, and hemorrhages overlying a normal pulp, 3 and 15 days postoperatively. A dense collagen-

FIG 3. Pulp exposure treated with Oycal for 29 days. A, Incomplete bridging of the exposure site with severely inflamed pulp (hematoxy- lin-eosin; original magnification x 16). B, Microbial colonies localized in the area outlined in A (Brown-Brenn; original magnification x 400).

594 Tziafas Journal of Endodontics

FIG 4. Pulp exposure treated with calcium hydroxide powder-saline paste for 29 days. A, Complete bridging of the exposure site overlay- ing normal pulp, with only hyperemic blood vessels (hematoxylin- eosin; original magnification x 16). B, A few bacteria localized in the area outlined in A (Brown-Brenn; original magnification x 400).

rich connective tissue was observed beneath the exposure site 29 days after the placing of Teflon as capping material (Fig. 5). No evidence of hard tissue formation was found in any tooth capped with Teflon.

Presence of Bacteria

Microorganisms were not detected in the histologically normal pulps of the six unoperated control teeth; bacteria

FIG 5. Pulp exposure capped with Teflon for 29 days. Collagen-rich connective tissue beneath the exposure site overlaying normal pulp (hematoxylin-eosin; original magnification x 16).

were only seen within the pulpal blood vessels. No microor- ganisms were found in the treated control teeth that had been extracted before the induction of bacteremia.

Bacteria were totally absence in three pulps 29 days after they had been capped with Teflon. In all of the remaining specimens of the experimental group, the pulp was infected by colonies of gram-positive cocci. The microorganisms were mainly localized in the pulp tissue close to the calcified barrier or in the reaction zone at some distance from the wound surface. Teeth with a more extensive reaction zone demon- strated a larger area of infection. A few scattered microorga- nisms were also identified in the underlying pulp tissue, as well as within the pulpal blood vessels in some specimens.

The greatest density of bacteria was found in the pulps treated for 3 days. No difference was observed in the micro- biological condition of the pulps between the teeth treated with Dycal and those treated with calcium hydroxide powder- saline paste.

DISCUSSION

Bacterial staining of the tissue sections in this study revealed the presence of coccal colonies in all the calcium hydroxide- treated teeth 24 h after induction of bacteremia. An experi- mentally exposed pulp treated with calcium hydroxide mate- rials may be infected by microorganisms by several routes: (a) by leaking of bacteria around the cavity restoration, (b) by contamination of the wound surface by the oral bacteria at the time of operation, (c) from a periodontal pocket, and (d) by localization from the blood stream. It was characteristically observed in the present experiment that the pulpal infection in all specimens was not accompanied by presence of gram- positive or negative bacteria along the cavity walls and within the cut dentinal tubules. This later criterion has been exten- sively used in previous pulp-capping experiments to indicate that the pulpal reactions may not be attributed to the presence of oral microorganisms (19, 22-26). Thus, pulp infection from

Vol. 15, No. 12, December 1989

oral microorganisms leaking around the cavity restorations can be ruled out. Also, the presence of bacteria in these specimens may not be attributed to contamination at the time of operation; bacteria are destroyed by the high pH of the calcium hydroxide-containing materials (27, 28). Cox et al. (24) did not observe pulpal infection 5 wk after capping of pulp exposures which had been left open to the oral environ- ment for 1 and 24 h. Even after 2-day experimental infection of pulp exposures before their treatment with calcium hy- droxide, Brannstr/3m et al. (29) found that the extension of the bacterial zone in the pulp was clearly limited to the area close to the exposure site; and, in all cases, bacteria were seen along the cavity wails. So, since periodontally diseased teeth were not used in this study, it is not unreasonable to assume that the microorganisms which were identified in the treated tissues had reached the pulp sites via the hematogenous route. The identity of the detected microorganisms cannot be deter- mined with histobacterial stains. However, the total absence of bacterial staining reactions in tissue sections of calcium hydroxide-treated teeth which had been extracted before the induction of bacteremia indicates that the microorganisms seen in pulps of the experimental group are those which had been intravenously injected.

The absence of bacteria in the histologically normal pulps of the unoperated control teeth and in pulps which had been capped with Teflon for 29 days indicates that uninflamed pulps or fully recovered pulps do not attract microorganisms through the anachoretic effect. This finding is in agreement with previous observations (6-10). Based on this, the localiza- tion of blood-borne microorganisms in the pulps which ex- hibited complete hard tissue barrier formation corroborates the previous conclusions that bridging of the exposure site does not indicate pulp repair (13-14, 23, 30).

The data collected in this study support the conjecture that during a bacteremia, the early tissue reactions after pulp- capping treatment with calcium hydroxide attract bacteria to the pulp. The localization of blood-borne microorganisms in the interstitial spaces of the pulp may be attributed to the destruction of the pulp vessels and/or the increased vascular permeability resulting from inflammation and from chemical injury to the vascular endothelium produced by the calcium hydroxide-released hydroxyl ions. Also, it was demonstrated that pulp infection occurred in all treated teeth with Ca(OH)~ based materials. The intensity of this pulp infection was dependent on the tissue response to the capping treatment. Bacteria were not detected in only three out of four Teflon- treated teeth. These findings indicate that the chemical effect of calcium hydroxide might be considered as a factor respon- sible for the infiltration of blood-borne bacteria to the reacting pulp tissue. In a further experiment the question of whether the capping of teeth with other materials producing lower chemical injury to the underlying tissue (calcium phosphate biomaterials) could result in a different pulpal response to circulating bacteria will be investigated.

Dr. Tziafas is a lecturer in the Department of Dental Pathology and Thera- peutics, School of Dentistry, Aristotle University, Thessaloniki, Greece. Address

Anachoresis in Capped Pulps 595

requests for reprints to Dimitdos Tziafas, Department of Dental Pathology and Therapeutics, School of Dentistry, Aristotle University, Thessaloniki, Greece.

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