28/08/2018 0 Σχόλια
Determination of endotoxins in caries: association with pulpal pain
M. G. Khabbaz, P. L. Anastasiadis & S. N. Sykaras Department of Endodontics, University of Athens, Dental School, Athens, Greece
Abstract Khabbaz MG, Anastasiadis PL, Sykaras SN. Determination of endotoxins in caries: association with pulpal pain. International Endodontic Journal, 33, 132±137, 2000.
Aim The aims of this study were: (i) to determine the presence or absence of endotoxins in the superficial and deep layers of carious lesions of symptomatic and asymptomatic teeth with vital pulps; (ii) to quantify the amount of endotoxin present; and (iii) to associate the presence of endotoxins with the acute pulpal pain derived from the irreversible pulpitis.
Methodology Two specimens of carious dentine were taken under aseptic conditions from symptomatic teeth with irreversible pulpitis (n = 9) and asymptomatic teeth with reversible pulpitis (n = 11). The first specimen was taken from a layer of superficial caries and the second from a deeper one. Sound dentine was also collected from intact teeth without restoration and used as a noncarious control group (n = 4). During the patient sampling procedure an effort was made to collect an equal quantity of caries and sound dentine in all cases (approximately 6 mg).
The extraction of endotoxins was performed using the Phenol-water method. The assay and quantitative determination of endotoxins was performed by the Quantitative Chromogenic test using Limulus Lysate. Data were analysed statistically using either independent or paired t-tests.
Results The results indicated that endotoxins were present in the superficial and deep layers of caries of all symptomatic teeth with irreversible pulpitis (0.15078 and 0.12111 ng mL-¹, respectively), with significantly greater amount (P < 0.01) in the superficial compared to the deep layer. Endotoxins were found in superficial and deep layer of caries of all asymptomatic teeth with reversible pulpitis (0.12091 and 0.07163 ng mL-¹, respectively), with significantly greater amounts (P < 0.001) in the superficial compared to the deep layer.
The results also demonstrated that significantly greater concentrations (P < 0.005) of endotoxins were present in the superficial carious layer of symptomatic compared with asymptomatic teeth (0.15078 and 0.12091 ng mL-¹, respectively). Likewise, significantly greater amounts (P < 0.001) of endotoxins were present in the deep carious layer of symptomatic compared with asymptomatic teeth (0.12111 and 0.07163 ng mL-¹, respectively). In sound dentine no endotoxins were detected.
Conclusions This study demonstrates that endotoxins are present in carious lesions of symptomatic and asymptomatic teeth. The amount of endotoxin was significantly greater in the superficial compared to the deep layer of carious dentine. More endotoxins are present in caries of painful teeth compared with those without symptoms.
Keywords: caries, endotoxin, pulpal pain.
Introduction Endotoxins are the lipopolysaccharide complex that constitute the cell wall component of Gram-negative bacteria. Endotoxins can be either secreted in vesicles by growing organisms or released into the environment after cell death. They are capable of initiating various biological responses such as complement activation, fever induction, macrophage activation, cytotoxicity and bone resorption (Davis et al. 1973, McKane & Kandel 1986).
It is well established that microorganisms are implicated in the aetiology of dental caries. In animals, many microbial strains have been shown to be capable of inducing caries (Orland et al. 1954, 1955, Edwardsson 1986). In humans the presence of grampositive (Streptococci, Lactobacilli, Actinomyces) and gram-negative (Cocci, Bacilli, Fusiforms, Bacteroides) microorganisms have been reported in carious dentine (Bibby & Hine 1938, Roth 1957, Hoshino 1985, Hahn et al. 1991, Schupbach et al. 1996).
Sarnat & Massler (1965) in an electron microscopic study emphasized the differentiation of two distinct layers in active carious lesions in humans: the bacteria rich layer (infected zone) with necrotic dentine and the deeper bacteria free layer where decalcification was present. Seltzer & Bender (1990) concurred with this view and reported that in the superficial layer irreversible denaturation of dentine and microbial infection existed whilst in the deeper layer the denaturation was reversible and without infection.
Nevertheless, many studies (Edwardsson 1974, Hoshino 1985, Hahn et al. 1991) have reported that the deep layers of carious dentine contain microorganisms. In some cases anaerobic bacteria were isolated from deep cavities even after the application of Ca(OH)2 for 6±9 months (Bjorndal et al. 1995).
Although many investigations have been carried out on the composition and genera of bacteria of carious dentine, little information is yet available regarding the existence of endotoxins. This information is important in understanding the role of endotoxins in mediating pulpal pain and/or inflammation.
The aims of this research were: (i) to determine the presence or absence of endotoxins in the superficial and deep layers of carious lesions of symptomatic and asymptomatic teeth diagnosed as irreversible and reversible pulpitis, respectively; (ii) to quantify the amount of endotoxin present; and (iii) to associate the presence of endotoxins with the acute pulpal pain derived from the irreversible pulpitis.
Materials and methods Patient selection Twenty-four teeth diagnosed as having irreversible and reversible pulpitis (nine molars and 15 premolars) from 21 patients (12-63 years old) who presented at the Clinic of Conservative Dentistry at the Dental School of the University of Athens, were included in this study. The teeth were divided into three groups, 1-Symptomatic carious (n = 9), 2-Asymptomatic carious (n = 11) and 3-Sound noncarious (control group, n 4). All patients agreed to participate in the study.
Clinical features
The following features were noted for each patient: age, gender, tooth, pulp vitality (electrical and thermal), nature of pain (duration, diffusion, sharp or dull), history of previous cavity preparation and local dentine-medication, periodontal status and radiographic findings.
All teeth in groups 1 and 2 had deep interproximal carious lesions in the crown without exposure of the dental pulp which was evaluated clinically and radiographically (at least 0.5 mm of dentine between pulpal space and caries seen in the radiograph). None of the teeth showed radiographic evidence of periapical inflammation. Group 1 (symptomatic carious) consisted of nine teeth with the clinical diagnosis of irreversible pulpitis. Teeth were placed into this group based on the following criteria: the teeth were currently causing spontaneous pain or had a recent history of causing severe pain to thermal stimuli which was diffused and lasted for minutes or for hours. All teeth responded to ice or electric pulp testing; a severe, prolonged pain reaction was elicited with ice. Group 2 (asymptomatic carious) consisted of 11 asymptomatic teeth with caries. Teeth were placed into this group based on the following criteria: no history of moderate or severe pulpal pain and no pain at the time of sampling. Clinical and radiographic examination determined the presence of caries without pulpal exposure, no signs of periapical pathosis and with normal to slight reaction to the vitality test. Group 3 (noncarious) consisted of four intact premolars of the maxilla from three patients aged 12-13 years that were scheduled to be extracted for orthodontic reasons. Teeth were placed into this group based on the following criteria: no history of pulpal pain; a clinical and radiographic examination that revealed no caries or restoration. Pulp testing revealed the pulp to be intact and healthy.
Teeth with a history of previous cavity preparation and local medication of dentine, or with periodontal pockets more than 3 mm, or with root caries, or which, after the excavation of the carious lesion, had an exposure of the pulp were not included in the study.
Patient sampling
All instruments used in this investigation were sterilized at 1808C for 4 h. All tubes used for the collection of specimens and experimental procedures were sterile (Whittaker Bioproducts, Inc. Walkersville, MD, USA.). The tips of the pipette were also sterile, ATP free and DNA free (Eurotips of Eppendorf, Hamburg, Germany).
After the diagnosis of reversible or irreversible pulpitis was made, local anaesthesia was administered and the appropriate tooth was thoroughly washed with air-water spray and dried. The rubber dam was placed and the outer surface of the tooth and the dam were disinfected with tincture of iodine and 70% ethyl alcohol. To avoid specimen contamination with dental plaque, less than 0.5 mm of carious dentine was removed, with a new and sterile bur on a low-speed motor without water spray.
The first specimen of carious dentine was taken with an excavator (No 82; Dentsply Maillefer, Ballaigues, Switzerland) from the superficial caries approximately 1 mm beneath the edges of the cavity and transfered into a sterile preweighed tube under aseptic conditions. Subsequently caries was removed from the cavity with a new sterile round bur on the low-speed motor until the deeper layer was exposed approximately 1.5±2 mm below the superficial layer. A second specimen was taken at this level and immediately transfered into a second tube.
To collect specimens from sound noncarious dentine, local anaesthesia was administered and rubber dam applied to the maxillary premolars. Initially, enamel was removed with a diamond without water spray. Afterwards, a sterile tube was placed in close proximity beneath the tooth and dentine was ground with a sterile round bur in the low-speed motor. During grinding sound dentinal chips were collected in the tube.
All tubes were reweighed with the specimens. During sampling an effort was made to collect an equal quantity of caries and sound dentine in all cases (approximately 6 mg).
Release of endotoxins
The extraction of endotoxins was performed using phenol-water (Westphal & Jann 1965). Three millilitres of a 65% phenol solution were added to the tube containing the specimen. The samples were shaken at room temperature for 1 min with a vortex mixer. After centrifugation at 3500 r.p.m. for 50 min, the supernatant was removed with a pipette and placed in a sterile tube for 10 min in a 608C water bath to disintegrate the proteins that may induce false positive results. Heating did not affect the endotoxin activity, since the endotoxin molecule is heat stable (Davis et al. 1973, McKane & Kandel 1986).
Measurement of endotoxins
The assay and quantitative determination of endotoxins was performed using a Limulus Lysate Test (Levin & Bang 1968). The Quantitative Chromogenic LAL-1000 (QCL-1000) was used as recommended by the manufacturer (Whittaker Biobroducts, Inc. Walkersville, MD, USA).
A phasmatophotometer (Spectronic 70, Bausch & Lomb, USA) was used at 405 nm to read the absorbance rate of endotoxins. The tubes were read by two persons in a blind test. A calibration curve was constructed initially and the concentration of endotoxins determined from this curve by linear regression.
Statistical analysis
Data on the presence or absence of severe and spontaneous pulpal pain, the quantitative determination of endotoxins in superficial and deep layers of caries were analysed statistically using the independent t-test and the paired t-test to compare the amount of endotoxins found in each layer of caries with the presence or absence of pulpal pain.
Results
Endotoxins were detected in all layers of caries of group 1 (symptomatic carious teeth) and group 2 (asymptomatic carious teeth). In group 3 (control noncarious teeth) endotoxins were not detected. The mean concentration of endotoxins in groups 1 and 2 is showed in Table 1.
Endotoxin concentration in symptomatic teeth
Endotoxins were detected in the superficial and deep layers of all symptomatic teeth. The endotoxin content in the superficial layer of carious dentine (average value 0.15078 ng mL-¹, SD = 0.02183) was significantly greater (P < 0.01) than in the deeper layer (average value 0.12111 ng mL-¹, SD = 0.01024).
Endotoxin concentration in asymptomatic teeth
Endotoxins were detected in superficial and in deep layers of all asymptomatic teeth. The endotoxin content in the superficial layer of carious dentine (average value 0.12091 ng mL-¹, SD = 0.01800) was significantly greater (P < 0.001) than in the deeper layer (average value 0.07163 ng mL-¹, SD = 0.01271).
Comparison of endotoxin concentration between symptomatic and asymptomatic teeth
The concentration of endotoxins in the superficial layer of symptomatic teeth (average value 0.15078 ng mL-¹, SD = 0.02183) was significantly greater (P < 0.005) than in asymptomatic teeth (average value 0.12091 ng mL-¹, SD = 0.01800) (Table 2).
The endotoxin concentration of deep layers in symptomatic teeth (average value 0.12111 ng mL-¹, SD = 0.01024) was significantly greater (P < 0.001) than in asymptomatic teeth (average value 0.07163 ng mL1 , SD = 0.01271) (Table 2).
Discussion
It is well documented, that oral microorganisms play an important role in the initiation and progression of caries and pulpal inflammation in human teeth (Kakehashi et al. 1965, Loesche & Syed 1973, Hoshino 1985, Hahn et al. 1991). Previous studies reported that there was no infection in the deep layer of carious dentine (Sarnat & Massler 1965, Wirthlin 1970). However, our results indicate that endotoxins are present not only in the superficial but also in the deep layers of caries. On the basis that endotoxin probably reflects the gram-negative microbial flora (Schein & Schilder 1975, Fine et al. 1992), these results confirm the findings of earlier reports that when soft dentine is left in deep cavities, microorganisms are present in most cases (Seltzer 1940, Shovelton 1968).
The results of this investigation demonstrated that the amount of endotoxin in superficial layers of carious dentine was significantly greater than in the deep layers. These results are in agreement with the results of Hoshino (1985) who found that after aerobic and anaerobic incubation of specimens taken from carious dentine, the number of microorganisms recovered was greater in shallow than in deeper layers. It seems that in superficial caries, where dentine is totally denatured, conditions are suitable for the multiplication of bacteria, whilst in deeper layers conditions may be different because of the reduced permeability of carious dentine (Miller & Massler 1962) or because of the differences in the oxygen concentration between the shallow and the deep layer of caries; this hypothesis has to be tested with further investigation.
Administration of endotoxin to experimental animals produces inflammation in the pulpal tissue (Warfvinge et al. 1985, Hirafuji & Shinoda 1994). When endotoxins were topically applied to freshly exposed dentine, Warfvinge et al. (1985) demonstrated that they were capable of provoking inflammatory cell migration in the dental pulp. Furthermore, when endotoxins were injected intravenously in the rat, Hirafuji & Shinoda (1994) demonstrated that they increased the production of prostaglandin I2 and thromboxane A2 in the dental pulp.
Endotoxins may evoke pain through activation of the Hageman factor that leads to the production of bradykinin, a potent pain mediator (Seltzer & Farber 1994). Similarly, a number of studies have reported that endotoxins possess neurotoxic properties acting on presynaptic nerve terminals (Penner & Bernheim 1960, Palmiero et al. 1962, Parnas et al. 1971). However, clinical symptoms were strongly associated with the presence of Bacteroides melaninogenicus (prevotella), an anaerobic gram-negative rod that produces endotoxin in the root canal of symptomatic nonvital teeth (Sundqvist 1976, Griffee et al. 1980, Gomes et al. 1994). Previous studies (Schein & Schilder 1975, Schonfeld et al. 1982, Horiba et al. 1991) found a correlation between bacterial endotoxins and pulpal or periradicular pain, because there was a higher endotoxin level in symptomatic teeth compared to those without symptoms.
In our study, endotoxin concentration in the superficial and deep layers of carious dentine was significantly greater in symptomatic teeth with irreversible pulpitis compared with asymptomatic teeth. Since the endotoxins have inflammatory and neurotoxic properties, the higher level of endotoxins in the caries of symptomatic teeth perhaps constitutes one factor amongst many others involved in the mechanism of pulpal pain.
To prove this hypothesis, penetration of endotoxin into dentinal tubules and their diffusion to the pulp through dentine must be proved. Application of crude bacterial extracts or specific bacterial components to exposed dentine can induce inflammation in the underlying pulp, indicating that bacterial products are probably capable of diffusing to the pulp (Bergenholtz 1977). Further evidence was provided when endotoxin was applied to the pulpal floor of class V cavities in monkeys (Warfvinge et al. 1985); accumulation of neutrophils in the pulp beneath exposed dentinal tubules suggested that endotoxin diffused into the pulp and activated the complement system, thus producing C5a, a chemotactic factor for neutrophils. Also Nissan et al. (1995), studying the ability of bacterial endotoxin to diffuse through human dentine, found that it was capable of passing through 0.5 mm of dentine. Despite these laboratory studies, the presence of endotoxins in the human dental pulp is yet to be proved.
The high molecular weight of endotoxins may be one factor that slows the passage of endotoxins through dentine (Segal et al. 1990). However, it has been well established that carious dentine is much less permeable than sound dentine (Miller & Massler 1962, Trowbridge 1981, Arends et al. 1995). The recovery of endotoxin in superficial and deep layers of caries and the association with the clinical symptoms despite the decreased permeability of the carious dentine is an important finding.
Conclusion
In conclusion, this study shows that: Endotoxins exist in caries of symptomatic and asymptomatic vital teeth. The amount of endotoxin is significantly greater in the superficial compared to the deep layer of carious dentine. More endotoxins are present in caries of painful teeth than in those without symptoms.
Acknowledgements
This study was supported by a grant from the University of Athens. The authors thank Ms Mantzarea K. for her technical support.
References
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endotoxins_and_pulpal_pain.pdf
Abstract Khabbaz MG, Anastasiadis PL, Sykaras SN. Determination of endotoxins in caries: association with pulpal pain. International Endodontic Journal, 33, 132±137, 2000.
Aim The aims of this study were: (i) to determine the presence or absence of endotoxins in the superficial and deep layers of carious lesions of symptomatic and asymptomatic teeth with vital pulps; (ii) to quantify the amount of endotoxin present; and (iii) to associate the presence of endotoxins with the acute pulpal pain derived from the irreversible pulpitis.
Methodology Two specimens of carious dentine were taken under aseptic conditions from symptomatic teeth with irreversible pulpitis (n = 9) and asymptomatic teeth with reversible pulpitis (n = 11). The first specimen was taken from a layer of superficial caries and the second from a deeper one. Sound dentine was also collected from intact teeth without restoration and used as a noncarious control group (n = 4). During the patient sampling procedure an effort was made to collect an equal quantity of caries and sound dentine in all cases (approximately 6 mg).
The extraction of endotoxins was performed using the Phenol-water method. The assay and quantitative determination of endotoxins was performed by the Quantitative Chromogenic test using Limulus Lysate. Data were analysed statistically using either independent or paired t-tests.
Results The results indicated that endotoxins were present in the superficial and deep layers of caries of all symptomatic teeth with irreversible pulpitis (0.15078 and 0.12111 ng mL-¹, respectively), with significantly greater amount (P < 0.01) in the superficial compared to the deep layer. Endotoxins were found in superficial and deep layer of caries of all asymptomatic teeth with reversible pulpitis (0.12091 and 0.07163 ng mL-¹, respectively), with significantly greater amounts (P < 0.001) in the superficial compared to the deep layer.
The results also demonstrated that significantly greater concentrations (P < 0.005) of endotoxins were present in the superficial carious layer of symptomatic compared with asymptomatic teeth (0.15078 and 0.12091 ng mL-¹, respectively). Likewise, significantly greater amounts (P < 0.001) of endotoxins were present in the deep carious layer of symptomatic compared with asymptomatic teeth (0.12111 and 0.07163 ng mL-¹, respectively). In sound dentine no endotoxins were detected.
Conclusions This study demonstrates that endotoxins are present in carious lesions of symptomatic and asymptomatic teeth. The amount of endotoxin was significantly greater in the superficial compared to the deep layer of carious dentine. More endotoxins are present in caries of painful teeth compared with those without symptoms.
Keywords: caries, endotoxin, pulpal pain.
Introduction Endotoxins are the lipopolysaccharide complex that constitute the cell wall component of Gram-negative bacteria. Endotoxins can be either secreted in vesicles by growing organisms or released into the environment after cell death. They are capable of initiating various biological responses such as complement activation, fever induction, macrophage activation, cytotoxicity and bone resorption (Davis et al. 1973, McKane & Kandel 1986).
It is well established that microorganisms are implicated in the aetiology of dental caries. In animals, many microbial strains have been shown to be capable of inducing caries (Orland et al. 1954, 1955, Edwardsson 1986). In humans the presence of grampositive (Streptococci, Lactobacilli, Actinomyces) and gram-negative (Cocci, Bacilli, Fusiforms, Bacteroides) microorganisms have been reported in carious dentine (Bibby & Hine 1938, Roth 1957, Hoshino 1985, Hahn et al. 1991, Schupbach et al. 1996).
Sarnat & Massler (1965) in an electron microscopic study emphasized the differentiation of two distinct layers in active carious lesions in humans: the bacteria rich layer (infected zone) with necrotic dentine and the deeper bacteria free layer where decalcification was present. Seltzer & Bender (1990) concurred with this view and reported that in the superficial layer irreversible denaturation of dentine and microbial infection existed whilst in the deeper layer the denaturation was reversible and without infection.
Nevertheless, many studies (Edwardsson 1974, Hoshino 1985, Hahn et al. 1991) have reported that the deep layers of carious dentine contain microorganisms. In some cases anaerobic bacteria were isolated from deep cavities even after the application of Ca(OH)2 for 6±9 months (Bjorndal et al. 1995).
Although many investigations have been carried out on the composition and genera of bacteria of carious dentine, little information is yet available regarding the existence of endotoxins. This information is important in understanding the role of endotoxins in mediating pulpal pain and/or inflammation.
The aims of this research were: (i) to determine the presence or absence of endotoxins in the superficial and deep layers of carious lesions of symptomatic and asymptomatic teeth diagnosed as irreversible and reversible pulpitis, respectively; (ii) to quantify the amount of endotoxin present; and (iii) to associate the presence of endotoxins with the acute pulpal pain derived from the irreversible pulpitis.
Materials and methods Patient selection Twenty-four teeth diagnosed as having irreversible and reversible pulpitis (nine molars and 15 premolars) from 21 patients (12-63 years old) who presented at the Clinic of Conservative Dentistry at the Dental School of the University of Athens, were included in this study. The teeth were divided into three groups, 1-Symptomatic carious (n = 9), 2-Asymptomatic carious (n = 11) and 3-Sound noncarious (control group, n 4). All patients agreed to participate in the study.
Clinical features
The following features were noted for each patient: age, gender, tooth, pulp vitality (electrical and thermal), nature of pain (duration, diffusion, sharp or dull), history of previous cavity preparation and local dentine-medication, periodontal status and radiographic findings.
All teeth in groups 1 and 2 had deep interproximal carious lesions in the crown without exposure of the dental pulp which was evaluated clinically and radiographically (at least 0.5 mm of dentine between pulpal space and caries seen in the radiograph). None of the teeth showed radiographic evidence of periapical inflammation. Group 1 (symptomatic carious) consisted of nine teeth with the clinical diagnosis of irreversible pulpitis. Teeth were placed into this group based on the following criteria: the teeth were currently causing spontaneous pain or had a recent history of causing severe pain to thermal stimuli which was diffused and lasted for minutes or for hours. All teeth responded to ice or electric pulp testing; a severe, prolonged pain reaction was elicited with ice. Group 2 (asymptomatic carious) consisted of 11 asymptomatic teeth with caries. Teeth were placed into this group based on the following criteria: no history of moderate or severe pulpal pain and no pain at the time of sampling. Clinical and radiographic examination determined the presence of caries without pulpal exposure, no signs of periapical pathosis and with normal to slight reaction to the vitality test. Group 3 (noncarious) consisted of four intact premolars of the maxilla from three patients aged 12-13 years that were scheduled to be extracted for orthodontic reasons. Teeth were placed into this group based on the following criteria: no history of pulpal pain; a clinical and radiographic examination that revealed no caries or restoration. Pulp testing revealed the pulp to be intact and healthy.
Teeth with a history of previous cavity preparation and local medication of dentine, or with periodontal pockets more than 3 mm, or with root caries, or which, after the excavation of the carious lesion, had an exposure of the pulp were not included in the study.
Patient sampling
All instruments used in this investigation were sterilized at 1808C for 4 h. All tubes used for the collection of specimens and experimental procedures were sterile (Whittaker Bioproducts, Inc. Walkersville, MD, USA.). The tips of the pipette were also sterile, ATP free and DNA free (Eurotips of Eppendorf, Hamburg, Germany).
After the diagnosis of reversible or irreversible pulpitis was made, local anaesthesia was administered and the appropriate tooth was thoroughly washed with air-water spray and dried. The rubber dam was placed and the outer surface of the tooth and the dam were disinfected with tincture of iodine and 70% ethyl alcohol. To avoid specimen contamination with dental plaque, less than 0.5 mm of carious dentine was removed, with a new and sterile bur on a low-speed motor without water spray.
The first specimen of carious dentine was taken with an excavator (No 82; Dentsply Maillefer, Ballaigues, Switzerland) from the superficial caries approximately 1 mm beneath the edges of the cavity and transfered into a sterile preweighed tube under aseptic conditions. Subsequently caries was removed from the cavity with a new sterile round bur on the low-speed motor until the deeper layer was exposed approximately 1.5±2 mm below the superficial layer. A second specimen was taken at this level and immediately transfered into a second tube.
To collect specimens from sound noncarious dentine, local anaesthesia was administered and rubber dam applied to the maxillary premolars. Initially, enamel was removed with a diamond without water spray. Afterwards, a sterile tube was placed in close proximity beneath the tooth and dentine was ground with a sterile round bur in the low-speed motor. During grinding sound dentinal chips were collected in the tube.
All tubes were reweighed with the specimens. During sampling an effort was made to collect an equal quantity of caries and sound dentine in all cases (approximately 6 mg).
Release of endotoxins
The extraction of endotoxins was performed using phenol-water (Westphal & Jann 1965). Three millilitres of a 65% phenol solution were added to the tube containing the specimen. The samples were shaken at room temperature for 1 min with a vortex mixer. After centrifugation at 3500 r.p.m. for 50 min, the supernatant was removed with a pipette and placed in a sterile tube for 10 min in a 608C water bath to disintegrate the proteins that may induce false positive results. Heating did not affect the endotoxin activity, since the endotoxin molecule is heat stable (Davis et al. 1973, McKane & Kandel 1986).
Measurement of endotoxins
The assay and quantitative determination of endotoxins was performed using a Limulus Lysate Test (Levin & Bang 1968). The Quantitative Chromogenic LAL-1000 (QCL-1000) was used as recommended by the manufacturer (Whittaker Biobroducts, Inc. Walkersville, MD, USA).
A phasmatophotometer (Spectronic 70, Bausch & Lomb, USA) was used at 405 nm to read the absorbance rate of endotoxins. The tubes were read by two persons in a blind test. A calibration curve was constructed initially and the concentration of endotoxins determined from this curve by linear regression.
Statistical analysis
Data on the presence or absence of severe and spontaneous pulpal pain, the quantitative determination of endotoxins in superficial and deep layers of caries were analysed statistically using the independent t-test and the paired t-test to compare the amount of endotoxins found in each layer of caries with the presence or absence of pulpal pain.
Results
Endotoxins were detected in all layers of caries of group 1 (symptomatic carious teeth) and group 2 (asymptomatic carious teeth). In group 3 (control noncarious teeth) endotoxins were not detected. The mean concentration of endotoxins in groups 1 and 2 is showed in Table 1.
Endotoxin concentration in symptomatic teeth
Endotoxins were detected in the superficial and deep layers of all symptomatic teeth. The endotoxin content in the superficial layer of carious dentine (average value 0.15078 ng mL-¹, SD = 0.02183) was significantly greater (P < 0.01) than in the deeper layer (average value 0.12111 ng mL-¹, SD = 0.01024).
Endotoxin concentration in asymptomatic teeth
Endotoxins were detected in superficial and in deep layers of all asymptomatic teeth. The endotoxin content in the superficial layer of carious dentine (average value 0.12091 ng mL-¹, SD = 0.01800) was significantly greater (P < 0.001) than in the deeper layer (average value 0.07163 ng mL-¹, SD = 0.01271).
Comparison of endotoxin concentration between symptomatic and asymptomatic teeth
The concentration of endotoxins in the superficial layer of symptomatic teeth (average value 0.15078 ng mL-¹, SD = 0.02183) was significantly greater (P < 0.005) than in asymptomatic teeth (average value 0.12091 ng mL-¹, SD = 0.01800) (Table 2).
The endotoxin concentration of deep layers in symptomatic teeth (average value 0.12111 ng mL-¹, SD = 0.01024) was significantly greater (P < 0.001) than in asymptomatic teeth (average value 0.07163 ng mL1 , SD = 0.01271) (Table 2).
Discussion
It is well documented, that oral microorganisms play an important role in the initiation and progression of caries and pulpal inflammation in human teeth (Kakehashi et al. 1965, Loesche & Syed 1973, Hoshino 1985, Hahn et al. 1991). Previous studies reported that there was no infection in the deep layer of carious dentine (Sarnat & Massler 1965, Wirthlin 1970). However, our results indicate that endotoxins are present not only in the superficial but also in the deep layers of caries. On the basis that endotoxin probably reflects the gram-negative microbial flora (Schein & Schilder 1975, Fine et al. 1992), these results confirm the findings of earlier reports that when soft dentine is left in deep cavities, microorganisms are present in most cases (Seltzer 1940, Shovelton 1968).
The results of this investigation demonstrated that the amount of endotoxin in superficial layers of carious dentine was significantly greater than in the deep layers. These results are in agreement with the results of Hoshino (1985) who found that after aerobic and anaerobic incubation of specimens taken from carious dentine, the number of microorganisms recovered was greater in shallow than in deeper layers. It seems that in superficial caries, where dentine is totally denatured, conditions are suitable for the multiplication of bacteria, whilst in deeper layers conditions may be different because of the reduced permeability of carious dentine (Miller & Massler 1962) or because of the differences in the oxygen concentration between the shallow and the deep layer of caries; this hypothesis has to be tested with further investigation.
Administration of endotoxin to experimental animals produces inflammation in the pulpal tissue (Warfvinge et al. 1985, Hirafuji & Shinoda 1994). When endotoxins were topically applied to freshly exposed dentine, Warfvinge et al. (1985) demonstrated that they were capable of provoking inflammatory cell migration in the dental pulp. Furthermore, when endotoxins were injected intravenously in the rat, Hirafuji & Shinoda (1994) demonstrated that they increased the production of prostaglandin I2 and thromboxane A2 in the dental pulp.
Endotoxins may evoke pain through activation of the Hageman factor that leads to the production of bradykinin, a potent pain mediator (Seltzer & Farber 1994). Similarly, a number of studies have reported that endotoxins possess neurotoxic properties acting on presynaptic nerve terminals (Penner & Bernheim 1960, Palmiero et al. 1962, Parnas et al. 1971). However, clinical symptoms were strongly associated with the presence of Bacteroides melaninogenicus (prevotella), an anaerobic gram-negative rod that produces endotoxin in the root canal of symptomatic nonvital teeth (Sundqvist 1976, Griffee et al. 1980, Gomes et al. 1994). Previous studies (Schein & Schilder 1975, Schonfeld et al. 1982, Horiba et al. 1991) found a correlation between bacterial endotoxins and pulpal or periradicular pain, because there was a higher endotoxin level in symptomatic teeth compared to those without symptoms.
In our study, endotoxin concentration in the superficial and deep layers of carious dentine was significantly greater in symptomatic teeth with irreversible pulpitis compared with asymptomatic teeth. Since the endotoxins have inflammatory and neurotoxic properties, the higher level of endotoxins in the caries of symptomatic teeth perhaps constitutes one factor amongst many others involved in the mechanism of pulpal pain.
To prove this hypothesis, penetration of endotoxin into dentinal tubules and their diffusion to the pulp through dentine must be proved. Application of crude bacterial extracts or specific bacterial components to exposed dentine can induce inflammation in the underlying pulp, indicating that bacterial products are probably capable of diffusing to the pulp (Bergenholtz 1977). Further evidence was provided when endotoxin was applied to the pulpal floor of class V cavities in monkeys (Warfvinge et al. 1985); accumulation of neutrophils in the pulp beneath exposed dentinal tubules suggested that endotoxin diffused into the pulp and activated the complement system, thus producing C5a, a chemotactic factor for neutrophils. Also Nissan et al. (1995), studying the ability of bacterial endotoxin to diffuse through human dentine, found that it was capable of passing through 0.5 mm of dentine. Despite these laboratory studies, the presence of endotoxins in the human dental pulp is yet to be proved.
The high molecular weight of endotoxins may be one factor that slows the passage of endotoxins through dentine (Segal et al. 1990). However, it has been well established that carious dentine is much less permeable than sound dentine (Miller & Massler 1962, Trowbridge 1981, Arends et al. 1995). The recovery of endotoxin in superficial and deep layers of caries and the association with the clinical symptoms despite the decreased permeability of the carious dentine is an important finding.
Conclusion
In conclusion, this study shows that: Endotoxins exist in caries of symptomatic and asymptomatic vital teeth. The amount of endotoxin is significantly greater in the superficial compared to the deep layer of carious dentine. More endotoxins are present in caries of painful teeth than in those without symptoms.
Acknowledgements
This study was supported by a grant from the University of Athens. The authors thank Ms Mantzarea K. for her technical support.
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