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W.R. Moorer, S.K. Thoden van Velzen, and P.R. Wesselink, Amsterdam, The Netherlands

DEPARTMENT OF CARIOLOGY, ENDODONTOLOGY AND PEDODOhTOLOGY, FACULTY OF DENTISTRY. THE UNIVERSITY OF AMSTERDAM

Short-term and semi-long-term tissue reactions to a mixture of “endodontopathic” bacteria are described. The bacteria are enclosed in experimental implant tubes simulating some aspects of the infected dental pulp. Localized abscesses develop in response to mixed bacterial contents of the implants. No abscess formation occurred with pure cultures (that is, either component of the mixture). Pathogenic inocula attract leukocytes to the adjacent tissues, which become inflamed and cause influx of leukocytes into the lumen of the implant proper. (ORAL SURG. ORAL MED. ORAL PATHOL. 59:642-646, 1985)

ixed anaerobic infections of the pulp seem to be the primary cause of periapical pathosis in human beings’-3 as well as in experiments with nonhuman primates.4-6 Special methods for the sampling and culturing of anaerobic bacteria are needed in order that we may study the etiologic and microbial aspects of periapical disease. Because of the obvious ethical and financial implications of intracanal test- ing in primates, several experimental methods involv- ing implants in small animals have evolved for the testing of etiologic agents or the evaluation of dental materials. One of these, the subcutaneous implanta- tion of tubelike chambers, became one of the recom- mended standard procedures for dental-materials testing.7 Our laboratory tested several modifications of such tubelike implants.8 Refinement of that model to a root-canal-simulating device that mediated the slow release of fluids9 or bacterial suspensions’0 was made. In this model, the effect of the trauma due to the implantation procedure proper was avoided by filling the implant in situ several weeks after the actual implantation had taken place and after repair and reorganization of the tissues around the empty subcutaneous chamber.

It was the purpose of the present study to examine the short and semi-long-term tissue response to several “endodontopathic” bacteria enclosed in our implant. Special attention was paid to the supposedly

642

abcessogenic” properties of a common anaerobic member of the oral flora.

MATERIALS AND METHODS Animals and implant chambers

Twenty rabbits between 6 and 8 months of age were used in this study. They were F, hybrids of the Alaska and Vienna White strains, housed and cared for in individual cages at the Central Institute of the Blood Transfusion Service, Amsterdam, The Neth- erlands. The implant chambers were polyethylene tubes (high-pressure, low-density polyethylene, with a molecular weight of about 22,000, Talas, Ommen, The Netherlands) with an inside diameter of 3.5 mm and an outside diameter of 4.5 mm. Sections 45 mm in length were cut, after which one wall of the resulting cylinder was perforated radially at a dis- tance of 10 mm from one end by piercing with a No. 45 root canal reamer and rotating this instrument several times. The perforation was examined under a dissecting microscope. Only tubes in which the perforation had a diameter of 0.42 ?Z 0.03 mm were used. To close each tube at both ends, each end was dipped in melted casting wax.

implantation

A two-stage procedure of implantation and subse- quent filling of the tubes was described earlier.9,‘2 In

Volume 59 Abscess formation induced with endodontopathic bacteria 643 Number 6

short, after induction of anesthesia, two incision areas per animal (approximately 5 cm from each side of the vertebral column and at equal distance from the forelegs and hind legs) were painted witlh iodine, after which a subcutaneous space was created to accommodate the empty implant, the perforated end of which was directed toward the head of the rabbit. After suturing of the incision, the animals were kept 3 weeks for repair of the operation trauma. Then anesthesia was induced and a small incision was made over the caudal end of the implant, the tissue capsule that had formed over the waxed end was split, and the wax was pierced with a hot injection needle. The experimental fluids or bacterial suspen- sions were then delivered into the implant by means of a syringe. A volume of approximately 0.4 ml filled the implant almost completely. Subsequently, the waxed end was closed again with a warm instrument and the incision was sutured. Aseptic principles were carefully followed.

Bacteria

Because of the frequent association of periapical involvement and oral black-pigmented Bacteroides species and streptococci in the infected pulp space,‘-3.‘3 these organisms were selected for the experiments. .Bacteroides gingivalis strain H 185? a well-characterized periodontitis isolate, and Strepto- coccus mitis strain H325, a root canal isolate, were obtained from Dr. T.J.M. van Steenbergen, Oral Microbiology Department, Free University, Amster- dam. Pure cultures of the bacteria in supplemented (hemin, vitamin K1, and yeast extract) brain-heart infusion (Difco) broth, were grown in an anaerobic glove box by incubation for 4 days at 36°C. Experi- mental inocula for the implants consisted of 10.4 ml of the fully grown (>108 colony-forming units per milliliter) suspensions proper and of a 1: 1 mixture of both cultures. Uninoculated broth, a heated (10 minutes at 80°C) mixed bacterial suspension, as well as physiologic saline solution served as controls. The cultures and controls were transported anaerobically to the anima.1 quarters in capped, septum-closed bottles for immediate delivery into the implants by syringe and needle. In vitro tests for purit.y of the cultures and for sterility of the controls were made from unused aliquots from the bottles, which were transported back t.o the anaerobic glove box. At the end of the experimental periods, the contents of the implants were carefully aspirated by reversal of the filling procedure. The fluids were inspected by phase-contrast microscopy for the presence of leuko- cytes and pus. A.nother aliquot was cultured for viability testing of the remaining inoculum.

Table I. Design of the experiment: Number of implant/tissue specimens suitable for evaluation

Specimens removed after

2 wk 5 wk 13 wk

Control: Uninoculated 2 2 2 broth

Control: Saline 2 2 2 Experimental: S.mitis 2 2* It

culture Experimental: B.gingivalis 2 4 2

culture Experimental: Mixed 2 4 4

S.mitis/B.gingivalis Control: Heated mixed 2

culture

*One animal died with symptoms of a severe cold. tone implant excluded because of infection of the operation wound.

Protocol

The twenty rabbits were divided into three control and three experimental groups. Each animal received two empty implants; both were filled 3 weeks later with an identical inoculum (or control fluid) in order to avoid any possible (immunologic) interference or carry-over of infectious material from otherwise dissimilar experimental fluids applied to one animal. After the filling procedure, the animals were left untreated for experimental periods of 2, 5, or 13 weeks. Table I shows the experimental groups and the number of implants available for analysis. At the end of the experimental periods (for example, 5, 8, and 16 weeks after the implantation), the contents of the implants were carefully aspirated, after which the tubes with their surrounding tissues were excised.

Histology

The tube-tissue samples were fixed immediately in a mixture of 2% paraformaldehyde and 2.5% gluta- raldehyde in 0.1 M phosphate buffer at pH 7.2. After 6 hours of fixation, the tubes were carefully removed from each specimen, the position of the communica- ting hole was noted, and superfluous tissue was cut off. Further fixation, routine histologic processing, and enclosure in paraffin were followed by the cutting of 6 pm thick sections perpendicular to the long axis of tlte cavity that had contained the implant. Every fiftieth section was mounted, num- bered, and stained with hematoxylin and eosin. The slides were examined and their features were noted. The lumen in the sections that had contained the implant was slightly oval as a result of compression during the cutting of the tissue block and/or because

44 Moorer, Thoden van Velzen and Wesselink Oral Surg. .kne. 1985

gable II. Contents of the implants: Viability of the remaining bacteria and presence of leukocytes/pus after the three experimental periods

Viable bacteria Leukocytes/pus

Experimental periods (wk)

Inocuium 2 5 is Z/5 13

Uninoculated broth - - - - - - Saline - -- -- -

S.mitis ++ - - - -

B.gingivalis ++ -,I* - ++ i-f - Mixed cultures ++ ++ +* ++ + --/+a Heated mix - +

*See text for explanation.

of a tilt from the ideal perpendicular orientation of cutting. A few tissue sections were torn or distorted, but in all specimens good preservation of the former tissue-implant interfaces was obtained. From each series of slides taken from a tissue specimen, two or three slides representative of that specimen were coded. The randomized sample slides were screened for gross histologic appearance: extension and nature of the inflammatory infiltrates, local vascular reac- tion, and presence of abscesses and of tissue necro- sis.

ESULTS aeteriology

After the experimental period of 2 weeks, large amounts of viable bacteria were recovered from all experimental implants. After the S-week period, limited numbers of S. mitis were recovered from the S. mitis-filled implants, indicating diminishing via- bility of the inoculum. No 5’. mitis (that is, fewer than 100 colony-forming units per milliliter of implantation fluid) were recovered from the one S. mitis implant after 13 weeks. B. gingivalis was recovered in moderate amounts from two of the four

. gingivalis implants after 5 weeks. No viable bacteria were found after 13 weeks. However, from the implants that received the mixed cultures, both organisms (except B. gingivalis from one implant after 13 weeks) were recovered following all three experimental periods. Thus, the duration of the viable period of either bacterial component of the mixed cultures seems to be extended as compared to the viable period of the pure cultures (Table II). In no instances were bacteria found in the controls.

Leukocytes and pus in the implants

Phase-contrast inspection of the contents of the implants after the experimental periods revealed that

Table 111. Gross histologic features of tbe tissues surrounding the implants: Extension of tbe inflam- matory infiltrates and abscess formation

r

Extension* Abscess*

Experimental periods (wk) /

Inocuium 2 5 j 13 12 5 13

Uninoculated broth - - - - - - Saline - -- --

S.mitis - - -- --

Kgingivalis + + +- --*

Mixed cultures +/H- +++ +++ + i--k +* Heated mix - -

*see text for exP,lanation.

large numbers of leukocytes had gained access into several of the implants (Table II). Apparently, some viable cells with streaming protoplasm were present but were comparatively rare. Generally large clumps of 20 to 100 presumably dead cells, in addition to single (dead) leukocytes and cell fragments, domi- nated the microscopic fields.

Furthermore, bacteria, dead or alive, were seen in all of the experimental implants as well as in the implant that received the heated mixed culture.

Extension of the inflammatory response

Inflammatory infiltrates were not discernible around any of the control or of the S. mitis tubes. Thin fibrous capsules had formed around the implants, and the connective tissue appeared normal and well-organized. It was not possible to distinguish the coded sections originating from any period or the contents of these control or S. mitis-filled implants. Some granulocytes were seen occasionally in or very near the fibrous capsule. This seemed to occur especially in the 2-week samples but not in the 13-week specimens; however, it formed no firm basis for distinguishing the subgroups.

In contrast, local inflammatory responses charac- terized by dense accumulations of lymphocytes were encountered in all B. gingivalis specimens. In addi- tion, the 5-week sections contained some sparse neutrophils and the 13-w-eek sections showed scat- tered groups of neutrophils. In several cases, some necrotic tissue debris was present between the implant and the host tissue capsule.

The mixture of B. gingivalis and S. mitis evoked heavy and extensive inflammatory responses, charac- terized by numerous neutrophils in and around accumulations of lymphocytes. Foam cells were seen. The 13-week specimens showed the development of a “granuloma” at the position where the communicat-

Volume 59 Abscess formation induced with endodontopathic bacteria 645 Number 6

ing hole of the implant had contacted the host tissue. It was marked by dense strings of colla,gen with intertwined dense groups of lymphocytes. Scattered neutrophilic cells collected around those groups of lymphocytes. Islands of necrotic tissue remains were evident. In all the above cases (B. gingiualis and the B. gingivalis-S. mitis mixture) of inflammatory response, large amounts of monocytes (macrophages, fibroblasts) formed part of the infiltrate in the areas of inflammation. Vessels showed evidence of stasis, thrombi, diapedesis of leukocytes, and small hemor- rhages. Table III summarizes the gross histologic features.

Abscess formation

Neither the controls nor the S. mitis and B. gingivalis specimens showed evidence of abscess formation. Some necrotic tissue remains were seen in some of the B. gingivalis sections. Only one very small liquefaction focus was seen in one of the 13-week B. gingivalis specimens. However, the mix- ture of B. gingivalis and S. mitis seemed 1;o trigger abscess formation. Then, in areas with necrosis, evident abscess formation occurred in nine of the ten specimens. Large abscesses (those that occupied more than 10% of the volume of tissue adjacent to the site of the communicating hole of the implant) were noted in four out of four and two out of four 5-week and I. 3-week specimens, respectively (Table III).

DISCUSSIOM

Many variables are associated with the use of an actual experimental dental root canal.14 Therefore, we studied the reaction of connective tissue to the influx of endodontopathic bacteria by means of a simple subcutaneous implant. The implant tubes served as a bacterial depot, accommodating known cultures that were largely shielded from the host’s circulation. Yet they allowed a certain interchange that was shown to cause inflammatory reactions in the surrounding tissue, as well as an influx of leukocytes into the enclosed lumen of the implants. The prolonged viability of enclosed masses of bacte- ria within the host bears a certain resemblance to the shielded and chronic presence of bacteri.a in the necrotic (infected) pulp. Comparison of Tables II and III suggests that viability of bacteria per se need not be directly associated with an inflammatory response. Virulence of the bacteria and their prod- ucts, expressed at some time during their stay in the host, seems to be important. Thus, in our model, S. mitis is not virulent but B. gingivalis is. The mixed populations, however, evoke especially strong reac-

tions in the host. These resembled infectious immu- nologic granulomatous inflammation.15

The role of mixed anaerobic infections of the pulp in the development of periapical disease has been convincingly demonstrated.‘-6 Also, virulence testing of mixed bacterial isolates from the pulp has shown that bacterial synergy is essential in the establish- ment of acute infections.16

Our model demonstrates the (chronic) abscesso- genie properties of Bacteroides as a component of a mixed inoculum. Bacteroides organisms have come to be recognized as important pathogens of the human pulp. The use of sophisticated methods of anaerobic culturing techniques has been necessary to demonstrate this.lm3, 13* I73 l8 Interestingly, a new dis- tinct species of Bacteroides called Bacteroides en- dodontalis (formerly classified as B. gingivalis or B. asaccharolyticzds) was described recently.19 Al- though our material is limited, a tentative conclusion from the present work is that short-term as well as long-term tissue reactions to enclosed bacteria, in a model that simulates the dental root canal, occur. Abscess formation and the extension of the “granu- loma” appear to be dependent on the nature of the inoculum. It is not known whether the leukocytes that gain access into the implants that contain a pathogenic flora are capable of phagocytosis. The very presence of leukocytes attracted by chemotactic factors from the bacteria does, of course, indicate potential pathogenicity.

The influence of viability and the number of bacteria, as well as their shielded enclosement prop- er, are under current investigation.

The authors wish to thank Mr. A. van Hamersveld and Mr. H. Ziegeler from the Central Laboratory of the Blood Transfusion Service, Amsterdam, for their biotechnical work and care, and Mr. A.J. Lammens for histologic assistance.

REFERENCES

Sundqvist G: Bacteriological studies of necrotic dental pulps. Thesis, Umea University, Umea, Sweden, 1976, pp. 5-24. Griffee MB, Patterson SS, Miller CH, Kafrawy AH, Newton CW: The relationship of Bacteroides melaninogenicus to symptoms associated with pulpal necrosis. ORAL SURG ORAL MED ORAL PATHOL 50: 457-461, 1980. Keudell K, Powell G, Diemer R: Humoral antibodies to anaerobic bacteria isolated from patients with pulpal-periapi- cal disease. ORAL SURC ORAL MED ORAL PATHOL 53: 194- 197, 1982. Moller AJR, Fabricius L, DahlCn G, (ihman AE, Heyden G: Influence on periapical tissues of indigenous oral bacteria and necrotic pulp tissue in monkeys. Stand J Dent Res 89: 475-484, 1981. Fabricius L, DahlCn G, Helm SE, Miiller AJR: Influence of combinations of oral bacteria on periapical tissues of mon- keys. Stand J Dent Res 90:200-206, 1982. Dahlen G, Fabricius L, Heyden G, Holm SE, Miiller AJR:

646 Moorer, Thoden van Velzen md Wesselink Oral Surg. June, 1985

7

8.

9.

10.

11.

12.

13.

Apical periodontitis induced by selected bacterial strains in root canals of immunized and nonimmunized monkeys. Stand J Dent Res 90: 207-216, 1982. Federation Dentaire International, Commission on Dental Materials, Instruments, Equipment and Therapeutics: Rec- ommended standard practices for biological evaluation of dental materials. Int Dent J 30: 140-188, 1980. Makkes PC, Thoden van Velzen SK, Wesselink PR, De Greeve PCM: Polyethylene tubes as a model for the root canal. ORAL SURC ORAL MED ORAL PATHOL 44: 293-300, 1971. Wesselink PR, Van Aalst R, Van Es L, Van den Hooff A, Thoden van Velzen SK: Root canal-simulating experimental model for evaluation of tissue responses to chronic influx of foreign fluid substances. J Dent Res 61: 44-48, 1982. Moorer WR, Thoden van Velzen SK, Wesselink PR, Van Steenbergen TJM, De Graaff J: Bacteria in subcutaneous implants simulating the dental root canal. J Dent Res 62: 454 (Abstr. 48), 1983. Balows A: Anaerobic bacteria: role in disease, Springfield, Ill.. 1975. Charles C Thomas Publisher. Moorer WR, Thoden van Velzen SK, Wesseiink PR: Long- term tissue response to bacteria enclosed in a subcutaneous implant that simulates the infected dental root canal. Int Endodont J 17: 207-212, 1984. Zavistoski J, Dzink J, Onderdonk A, Bartlett J: Quantitative bacteriology of endodontic infections. ORAL SURG ORAL MED ORAL PATHOL 49: 171-174, 1980.

14.

15.

16.

17.

18.

19.

Rowe AHR: Problems of intra-canai testing of endodontic materials. Int Endod J 13: 96-103, 1980. Weissmann G (editor): The cell biology of inflammation, Amsterdam, 1980, Elsevier/North-Holland Biomedical Press, pp. 544-556. Sundqvist GK, Eckerbom MI, Larsson AP, Sjligren UT: Capacity of anaerobic bacteria from necrotic dental pulps to induce purulent infections. Infect Immun 25: 685-693, 1979. Dahlen G, Bergenholtz G: Endotoxic activity in teeth with necrotic pulps. J Dent Res 59: 1033-1040, 1980. Finegold SM: Taxonomy, enzymes, and clinical relevance of anaerobic bacteria Rev Infect Dis 1: 248-253, 1979. Van Steenbergen TJM, Van Winkelhoff AJ: Mayrand D, Grenier D, De Graaff J: Bacteroides endodontalis sp.nov., an asaccharolytic black-pigmented Bacteroides species from infected dental root canals. Int J Syst Bacterial 34: 118-120: 1984.

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