International Endodontic }ouma! (1992) 2 5 , 2 - 5
Bacterial invasion of non-exposed dental pulp E, HOSHINO, N, ANDO*. M. SATO& K. KOTA* Department of Oral Microbiology and 'Department ofOperative Dentistry and Endodontics, Niigata University School of Dentistry, Niigata, Japan
Summary
and tbe pulp is covered by clinically sound dentine, tbat small numbers of bacteria can still invade tbe dental pulp through only a few, specific dentinal tubules. Detection of such bacteria may be difficult, because tbe local defence cells may eliminate tbem easily, unless successive bacterial invasion occurs. In fact, it bas not been reported so far tbat bacteria can be isolated from pulp tissue covered by clinically sound dentine. However, tbe bacteria are probably obligate anaerobes that predominate in the deep layers of carious dentine (Hosbino 1985) and, if so, the adoption of anaerobic procedures makes it possible to detect tbe bacteria easily, Tbe aim of tbis study was to isolate and cbaracterize tbe bacteria tbat invaded the dentai pulp prior to clinical pulpitis.
Anaerobic procedures were adopted to demonstrate the early bacterial invasion of non-expos^ dental pulps, and to isolate and identify the bacteria. Of 19 freshly extracted teeth which originally exhibited deep dentinal lesions, clinical examination and electric pulp testing showed that nine erf'them had no pulpal esqiosure. Thus the pulps of diese teeth were covered by clinically sound dentine beneath the carious lesion. Bacteria were found to have invaded the pulps of six of these nine teeth. The predominant bacteria were obligate anaerobes belonging to the genera Eubaclerium, Propionibacterium and Actinomyces, Other obligate anaerobes were Lactobacillus, Peptostreptococcus, Veillonella and Streptococcus, The bacterial composition resembled that of the deep layers of dentinal lesions described Materials and methods previously, sugg^ting that the bacteria isolated in this study had passed through some individud dentinal Samples and isolation of micro-organisms tubules, to invade the dental pulp. A pulpal sample was takenfromeacb of 19 human teeth witb untreated occlusal carious lesions extending into Keywords: dental caries, dentat pulp, micro-organisms. tbe dentine but no clinicai evidence of open pulpai exposure. For a control, dental pulps were obtained from 24 sound teetb. All tbe teetb used in tbis study were Introduction permanent third molars with vital pulps. Immediately after extraction of each tooth, tbe root apex and tbe open It is generally accepted tbat bacteria do not invade tbe cavity were sealed with biue inlay wax under a gentle dental pulp if clinically sound dentine remains beneatb stream of nitrogen gas containing 5% CO^, The tooth tbe bottom of the carious lesion, altbougb bacterial surfaces were wiped for a few seconds witb gauze which invasion of tbe pulp through dentinai tubules appears to had been immersed in 70% ethanol solution. After being be induced by clinical dental procedures (Smulson & notched with a sterile diamond disc to facilitate splitting, Sieraski 1989). However, it bas been reported tbat bacthe tooth was immediately transferred into an anaerobic teria can be isolated from deep layers of dentinal lesions glove box (Model AZ-Hard, Hirasawa, Tokyo, Japan) wbere tbe dentine bas not been softened (Hosbino 1985, Ando & Hoshino 1990). In addition, microscopic obser- containing 80% nitrogen with 10% H^ and 10% CO^, While in the box, tbe tooth was then split horizontally vation bas revealed tbat bacteria advance into dentinal witb forceps, and tbe dental pulp remaining in the cortubules unevenly, i,e. deeper in some individual tubules onal half of the tootb was taken with sterilized dental (Reeves & Stanley 1966). Tbus it is possible, in teetb excavators and/or barbed broaches. Each of the pulpa! where there is no clinical evidence of pulpal exposure samples was weighed and suspended in sterilized 40 mM Correspondence: Dr Etsuro Hoshino, Department of Oral Microbiology, potassium pbosphate buffer (pH 7,0) at a concentration Niigata University School of Dentistry, Gakkochotiori 1, Niigata 9 5 1 , of 1 mg ml"', or in 1 mi ofthe same buffer, and dispersed japan.
Bacterialinvasion of non-exposed dental pulp with a glass homogenizer. After serial 10-fold dilution with the same buffer, 0.1-ml aiiquots of each dilution were spread over the surface of BHI-Blood (sheep) agar plates (Holdeman et al. 1977) and incubated in the anaerobic glove box at 37°C. Plates, media, buffer solutions and experimental instruments were kept in an anaerd>ic box for at least 24 h prior to use. For further inspection, all colonies (3-103) from each of the suitably diluted samples were isolated and identified after euiaerobic incubation. In this study, obligate anaerobes were defined as bacteria that only exhibited growth in an anaerobic glove box, and facultative bacteria as those that also exhibited growth in air with 30% COj (Hoshino 1985). It was confirmed at least three times during the identification procedure that the obligate anaerobes did not grow in air with 30% CO^.
Identification of bacterial genera Obligate anaerobes and some of the facultative bacteria that belong to the genera Actinomyces, Eubacterium, Lactobacillus and Propiombacterium, and anaerobic cocci were identified according to the VPl Anaerobe Laboratory Manual (Holdeman etal. 1977) supplemented with information from Bergey 's Manual of Systematic Bacteriology (Holdeman-Moore & Moore 1986, Moore & HoldemanMoore 1986). as described previously (Hoshino 1985, Ando & Hoshino 1990). Other facultative bacteria were identified according to Bergey's Manual of Systematic Bacteriology (Hardie 1986, Kandeler & Weiss 1986, Schaail986).
Examination for pulpal exposure After sampling the dental pulp, the coronal half of the tooth was taken out of the anaerobic glove box in order to measure the electrical resistance between the open occlusal cavity and the puip chamber. The measurement was carried out with a Endo-Meter {Onuki Iki Co., Tokyo. Japan) as described by Tomita (1962). Clinical pulpal exposure of molar teeth is predicted when the resistance is less than 15 Kfl (Tomita 1962), which corresponds to 30 (lA after conversion into electric current with the Endo-Meter. Thus, pulpal exposure is indicated when the Endo-Meter vaiue exceeds 30 jiA. Softened dentine was then removed in order to ascertain that the carious lesion did not extend to the dental pulp. Throughout this study, the removal of softened dentine was performed according to specific criteria (Sato & Fusayama 1976, Fukushima 1981), with the help of a 'caries detector'
Table 1. Bacterial recovery ik>ni dental pulp with or writfaout pulpal exposures Endo-Meter*
Sample
Before (HA)
Without pulpal exposure 1 2 3 4 5 6 7 S 9
20 29 — 12 9 25 — 26
With pulpal exposure 10 11 12 13 14 15 16 17 18 19
34 32 — 38 — 32 35 37 42 —
+
After MA)
8 24 24 8 14 26 25 22 22
CFUM
0 0
1.8x10' 3.0x10' 7.0x10' 2.1 X 10^ 3.3 X IW 5.8x10-!
0
3.0x10^ 1.0x10" 2.8x10^ 1.9x10' 1.2x10' >]0* >W >I0< >10
Bacterial invasion of non-exposed dental pulp E, HOSHINO, N, ANDO*. M. SATO& K. KOTA* Department of Oral Microbiology and 'Department ofOperative Dentistry and Endodontics, Niigata University School of Dentistry, Niigata, Japan
Summary
and tbe pulp is covered by clinically sound dentine, tbat small numbers of bacteria can still invade tbe dental pulp through only a few, specific dentinal tubules. Detection of such bacteria may be difficult, because tbe local defence cells may eliminate tbem easily, unless successive bacterial invasion occurs. In fact, it bas not been reported so far tbat bacteria can be isolated from pulp tissue covered by clinically sound dentine. However, tbe bacteria are probably obligate anaerobes that predominate in the deep layers of carious dentine (Hosbino 1985) and, if so, the adoption of anaerobic procedures makes it possible to detect tbe bacteria easily, Tbe aim of tbis study was to isolate and cbaracterize tbe bacteria tbat invaded the dentai pulp prior to clinical pulpitis.
Anaerobic procedures were adopted to demonstrate the early bacterial invasion of non-expos^ dental pulps, and to isolate and identify the bacteria. Of 19 freshly extracted teeth which originally exhibited deep dentinal lesions, clinical examination and electric pulp testing showed that nine erf'them had no pulpal esqiosure. Thus the pulps of diese teeth were covered by clinically sound dentine beneath the carious lesion. Bacteria were found to have invaded the pulps of six of these nine teeth. The predominant bacteria were obligate anaerobes belonging to the genera Eubaclerium, Propionibacterium and Actinomyces, Other obligate anaerobes were Lactobacillus, Peptostreptococcus, Veillonella and Streptococcus, The bacterial composition resembled that of the deep layers of dentinal lesions described Materials and methods previously, sugg^ting that the bacteria isolated in this study had passed through some individud dentinal Samples and isolation of micro-organisms tubules, to invade the dental pulp. A pulpal sample was takenfromeacb of 19 human teeth witb untreated occlusal carious lesions extending into Keywords: dental caries, dentat pulp, micro-organisms. tbe dentine but no clinicai evidence of open pulpai exposure. For a control, dental pulps were obtained from 24 sound teetb. All tbe teetb used in tbis study were Introduction permanent third molars with vital pulps. Immediately after extraction of each tooth, tbe root apex and tbe open It is generally accepted tbat bacteria do not invade tbe cavity were sealed with biue inlay wax under a gentle dental pulp if clinically sound dentine remains beneatb stream of nitrogen gas containing 5% CO^, The tooth tbe bottom of the carious lesion, altbougb bacterial surfaces were wiped for a few seconds witb gauze which invasion of tbe pulp through dentinai tubules appears to had been immersed in 70% ethanol solution. After being be induced by clinical dental procedures (Smulson & notched with a sterile diamond disc to facilitate splitting, Sieraski 1989). However, it bas been reported tbat bacthe tooth was immediately transferred into an anaerobic teria can be isolated from deep layers of dentinal lesions glove box (Model AZ-Hard, Hirasawa, Tokyo, Japan) wbere tbe dentine bas not been softened (Hosbino 1985, Ando & Hoshino 1990). In addition, microscopic obser- containing 80% nitrogen with 10% H^ and 10% CO^, While in the box, tbe tooth was then split horizontally vation bas revealed tbat bacteria advance into dentinal witb forceps, and tbe dental pulp remaining in the cortubules unevenly, i,e. deeper in some individual tubules onal half of the tootb was taken with sterilized dental (Reeves & Stanley 1966). Tbus it is possible, in teetb excavators and/or barbed broaches. Each of the pulpa! where there is no clinical evidence of pulpal exposure samples was weighed and suspended in sterilized 40 mM Correspondence: Dr Etsuro Hoshino, Department of Oral Microbiology, potassium pbosphate buffer (pH 7,0) at a concentration Niigata University School of Dentistry, Gakkochotiori 1, Niigata 9 5 1 , of 1 mg ml"', or in 1 mi ofthe same buffer, and dispersed japan.
Bacterialinvasion of non-exposed dental pulp with a glass homogenizer. After serial 10-fold dilution with the same buffer, 0.1-ml aiiquots of each dilution were spread over the surface of BHI-Blood (sheep) agar plates (Holdeman et al. 1977) and incubated in the anaerobic glove box at 37°C. Plates, media, buffer solutions and experimental instruments were kept in an anaerd>ic box for at least 24 h prior to use. For further inspection, all colonies (3-103) from each of the suitably diluted samples were isolated and identified after euiaerobic incubation. In this study, obligate anaerobes were defined as bacteria that only exhibited growth in an anaerobic glove box, and facultative bacteria as those that also exhibited growth in air with 30% COj (Hoshino 1985). It was confirmed at least three times during the identification procedure that the obligate anaerobes did not grow in air with 30% CO^.
Identification of bacterial genera Obligate anaerobes and some of the facultative bacteria that belong to the genera Actinomyces, Eubacterium, Lactobacillus and Propiombacterium, and anaerobic cocci were identified according to the VPl Anaerobe Laboratory Manual (Holdeman etal. 1977) supplemented with information from Bergey 's Manual of Systematic Bacteriology (Holdeman-Moore & Moore 1986, Moore & HoldemanMoore 1986). as described previously (Hoshino 1985, Ando & Hoshino 1990). Other facultative bacteria were identified according to Bergey's Manual of Systematic Bacteriology (Hardie 1986, Kandeler & Weiss 1986, Schaail986).
Examination for pulpal exposure After sampling the dental pulp, the coronal half of the tooth was taken out of the anaerobic glove box in order to measure the electrical resistance between the open occlusal cavity and the puip chamber. The measurement was carried out with a Endo-Meter {Onuki Iki Co., Tokyo. Japan) as described by Tomita (1962). Clinical pulpal exposure of molar teeth is predicted when the resistance is less than 15 Kfl (Tomita 1962), which corresponds to 30 (lA after conversion into electric current with the Endo-Meter. Thus, pulpal exposure is indicated when the Endo-Meter vaiue exceeds 30 jiA. Softened dentine was then removed in order to ascertain that the carious lesion did not extend to the dental pulp. Throughout this study, the removal of softened dentine was performed according to specific criteria (Sato & Fusayama 1976, Fukushima 1981), with the help of a 'caries detector'
Table 1. Bacterial recovery ik>ni dental pulp with or writfaout pulpal exposures Endo-Meter*
Sample
Before (HA)
Without pulpal exposure 1 2 3 4 5 6 7 S 9
20 29 — 12 9 25 — 26
With pulpal exposure 10 11 12 13 14 15 16 17 18 19
34 32 — 38 — 32 35 37 42 —
+
After MA)
8 24 24 8 14 26 25 22 22
CFUM
0 0
1.8x10' 3.0x10' 7.0x10' 2.1 X 10^ 3.3 X IW 5.8x10-!
0
3.0x10^ 1.0x10" 2.8x10^ 1.9x10' 1.2x10' >]0* >W >I0< >10
