J Dent Educ. 69(6): 633-638 2005
© 2005 American Dental Education Association
From the Students' Corner |
Fiber Optic Fluorescence Microprobe for Endodontic Diagnosis
Ani Sarkissian;
Ashley Nga Le
Key words: spectroscopy, fluorescence, endodontic therapy, root canal, dentin, pulp tissue, endotontic pathogens
Submitted for publication 11/18/04;
accepted 02/08/05
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Abstract
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Successful endodontic therapy requires total debridement as well as complete obturation of the root canal to the cemento-dentinal junction. The goal of this study was to investigate the feasibility of using quantitative fluorescence spectroscopy for the detection and localization of pathological dentin, pulpal remnants, and microorganisms within the root canal. Specific aims were to identify: 1) characteristic excitation/emission spectra for healthy dentin, decayed dentin, enamel, and pulp; 2) the potential of specific spectral data for differentiating between these tissues; and 3) the potential of spectral data for detecting the presence and identifying four common endodontic pathogens. Fluorescence spectra were determined in the tissues of permanent human teeth, extirpated healthy and necrotic pulps, and four endodontic pathogens. Excitation/emission spectra were collected at 366nm, 405nm, and 440nm excitation. Marked differences in spectral signatures between the different tissues under investigation were observed. We postulate that the differences in fluorescence spectra of decayed vs. healthy dentin are due to the loss of mineralized tissue components and increased organic presence and water in these tissues. Pulpal tissue showed distinctly different fluorescence spectra from healthy and decayed dentin, providing a basis for differentiating between tissue categories. Each bacterial species demonstrated distinct spectral emission patterns.
Root canal (endodontic) therapy is a procedure performed to restore the tooth to functionality after the onset of pulpal pathosis. The root canal must be cleansed of soft tissue, necrotic debris, and pathological dentin before filling. The elimination of bacterial contaminants is also desirable. During endodontic retreatment, any existing root canal filling material must also be removed completely.
Current techniques rely mainly on tactile feedback to assess the appropriate endpoint for filling and cleaning procedures and to evaluate completeness of root canal debridement and preparation. However, this approach offers no information on the persistence of localized areas of dentin pathology. Other than complex and time-consuming culture techniques, there exists no real-time capability for the determination of bacterial presence or identity within the root canal. Incomplete canal preparation may result in pain, patient treatment failure, or systemic disease. In one study by Ingle and Backland,1 involving fifty-five patients with apical periodontitis, the canals were cleaned and irrigated. Then the canals were cultured, with twenty-two teeth testing positive and thirty-three testing negative for bacterial presence prior to obturation. Of the thirty-three negative teeth, 94 percent showed entire root canal healing. The twenty-two teeth testing positive before filling had a decreased success rate of 68 percent, whereas fifteen of the twenty-two patients did not have complete healing of the root canal. Therefore, it is essential to completely remove bacteria in the canal prior to filling; otherwise, according to this study, the patient may have a 26 percent increased chance of treatment failure.
Fluorescence has been investigated in healthy versus decayed tooth substance as a tool for coronal caries detection.2 Using a 488nm laser light for excitation, fluorescence emissions differed sufficiently between carious and sound enamel to permit discrimination between the two. The fluorescence of enamel was consistently lower in areas of reduced mineral content.
A chairside battery-powered fluorescence device, DIAGNOdent, which aids in detecting caries, has been commercially developed. In this system, a 655nm diode laser light source is used to illuminate the teeth. The fluorescence is collected at 680nm.3–6 The 655nm wavelength selection in DIAGNOdent is still being evaluated for its strengths and weaknesses, including whether this device can detect demineralization in addition to decay. Since DIAGNOdent has a solid, inflexible probe body, it cannot be used within the root canal.
Infections in the root canal space are typically polymicrobial and almost invariably involve at least one anaerobic bacterial species.7–9 The bacterial species examined in this study were laboratory strains representative of the microorganisms found in the oral microbiota and associated with endodontic and other infectious diseases. Oral streptococci, such as Streptococcus oralis, are Gram-positive facultative species that are commonly isolated from orofacial and endodontic infections.7,10 In addition S. oralis is a member of the viridans streptococci that are causative agents of bacterial endocardititis.11 Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum are all Gram-negative oral anaerobic bacteria and common isolates from endodontic infections. P. gingivalis is frequently associated with periodontal disease; P. intermedia and F. nucleatum are part of the indigenous microbiota, but are greatly increased and implicated as pathogens in periodontal disease.12 P. intermedia and F. nucleatum are also commonly isolated from oral and non-oral mixed species infections.1,13,14
The goal of this study was to investigate the usefulness of quantitative fluorescence spectroscopy for the detection and localization of pathological dentin and pulpal remnants within the root canal. Specific aims were to 1) identify characteristic excitation/emission spectra for healthy dentin, decayed dentin, enamel, and pulp; 2) determine the potential of specific spectral data for differentiating between these tissues; and 3) evaluate the potential of spectral data for detecting the presence and determining the identity of four common endodontic pathogens.
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Materials and Methods
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All samples were collected and handled in accordance with UCI IRB 99*1245 safety code.
Eight extracted permanent human teeth were used for the hard tissue investigations. Five of these were clinically and radiographically healthy teeth, and three had root decay as determined by visual examination and probing. Prior to and during the study, samples were stored individually, wrapped in gauze, moistened with isotonic, sterile saline, and blotted dry before use, with time of storage and conditions of storage being kept constant for all samples. Universal precautions were taken when any samples were handled. After preparing conventional access to the pulp and after extirpation of pulpal tissues using a barbed broach, teeth were longitudinally bisected for spectral characterization (Figure 1
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The exact health status of each root canal measurement site with regard to infection/demineralization/caries was determined visually with an explorer, using radiographs and SEM, allowing precise correlation of dentinal health status with spectral traces. In each procedure, all fluorescence measurements on each tooth were repeated at least three times to allow statistical analysis of reproducibility of results.
Five healthy and five necrotic, freshly extirpated human pulp samples were obtained from the eight teeth, which were used to determine typical fluorescence spectra for these tissues. The bacterial strains used were Fusobacterium nucleatum subspecies nucleatum ATCC 23726, Prevotella intermedia ATCC 49046, Porphyromonas gingivalis ATCC 33277, and Streptococcus oralis 34. All strains were cultivated anaerobically in an atmosphere of 10 percent CO2, 10 percent H2, 80 percent N2, on Columbia agar with 5 percent sheep’s blood (Colorado Serum Co., Denver, CO). A comparably maintained but uninoculated agar plate was used for background analysis.
Excitation/emission spectra were obtained using a benchtop SPEX Fluorolog III spectrofluorometer with a magnification of 40x. Full excitation/ emission spectra were collected at 366nm 405nm 440nm excitation using bandpass 30, bandpass 20, and bandpass 40 filters respectively. Spectral acquisition time was 100ms.
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Results and Discussion
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Marked differences in spectral signatures between the different tissues under investigation were observed. Using excitation at 366nm, healthy dentin showed considerably higher fluorescence emission intensities at wavelengths of 440–500nm than decayed dentin, with a peak around 455–464nm (Figure 2). Characteristic healthy dentin and decayed dentin fluorescence spectra at excitations of 405nm and 440nm showed obvious differences (Figures 3 and 4). Healthy dentin had characteristic peaks at 494nm and 530nm after excitation at 405nm, whereas decayed dentin demonstrated characteristic peaks at 498nm, 533nm, 545nm, and 568nm. After excitation at 440nm, healthy and decayed dentin both showed an emission peak around 545nm, with decayed dentin exhibiting a secondary peak around 570nm. The differences in fluorescence spectra were attributed to the loss of mineralized tissue components and increased organic presence and water in decayed versus healthy dentin.
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Figure 2. Decayed dentin and healthy dentin at 366nm excitation
Note: X-axis is in measurements of nm, and Y-axis is arbitrary units.
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Figure 3. Excitation of decayed dentin and healthy dentin at 405nm
Note: X-axis is in measurements of nm, and Y-axis is arbitrary units.
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Figure 4. Excitation of decayed dentin and healthy dentin at 440nm
Note: X-axis is in measurements of nm, and Y-axis is arbitrary units.
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The fluorescence from pulp is substantially lower than the healthy dentin and decayed dentin fluorescence (Figure 5). The emission patterns for enamel are unique and can be differentiated from that of healthy dentin and decayed dentin patterns. The difference in emission spectrum was supported by Soret band absorption at approximately 415nm, perhaps reflecting characteristics of perfused vs. nonperfused pulp tissue. Further studies using larger numbers of specimens and optimal experimental protocols will be used to identify the most useful spectral criteria for clinical diagnostic purposes.
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Figure 5. Healthy dentin, decayed dentin, and pulp at 405nm excitation
Note: Pulp fluorescence is lower than healthy dentin and decayed dentin. X-axis is in measurements of nm, and Y-axis is arbitrary units.
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As these excitation/emission peaks correspond to those of collagen, it is likely that structural and pathology-related changes in collagen content are reflected in these results. The distinct fluorescence spectra of pulpal tissue vs. those of healthy dentin and decayed dentin provide a means of differentiating among the three tissue categories.
Common bacterial colonies found in infected root canals were excited at 405nm and 440nm (Figure 6). Each bacterial colony had its characteristic emission spectra, different from that of healthy dentin and decayed dentin (Figure 7). Spectral signatures from each bacterial species were quite distinct. P. gingivalis demonstrated high levels of fluorescence from 610 through 660nm with a marked peak at approx 630nm. P. intermedia also demonstrated distinct porphyrin-related fluorescence with distinct peaks at 630–640nm and 660–670nm. P. gingivalis and P. intermedia are both organisms that are part of a group of oral bacteria known as the "black-pigmented bacilli" because of their dark, porphyrin content-related appearance. S. oralis and F. nucleatum did not exhibit these peaks, with each organism demonstrating distinct spectra with characteristic peak patterns.
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Figure 6. Four common bacterial colonies: Porphyromonas gingivalis, Prevotella intermedia, Streptococcus oralis, and Fusobacterium nucleatum at 405nm excitation
Note: X-axis is in measurements of nm, and Y-axis is arbitrary units.
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Figure 7. Four bacterial colonies, decayed dentin, and healthy dentin at 405nm
Note: X-axis is in measurements of nm, and Y-axis is arbitrary units.
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In conclusion, this study demonstrated the potential for spectroscopic evaluation of root canal status throughout endodontic therapy. We envision the development of flexible fiber optic-based systems providing spatially resolved feedback on endodontic treatment needs and completeness to enhance the efficacy and success of root canal therapy. Our group has constructed a prototype fiberoptic probe system for further studies. Similar probes abound for other fiberoptic applications in medicine. They are compact, approximately the size of a pen, relatively easy and inexpensive to fabricate, and can be cold sterilized.
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Acknowledgments
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We would like to express our deepest gratitude to all of those who have dedicated so many hours to helping us complete this project. First, we would like to thank Dr. Arata Ebihara for helping us on the clinical details and relevance and Dr. Susan Haake for supplying us with the bacteria samples. We would also like to thank Romin Zorin and Tatiana Krasieva for their assistance with the use of the spectrometer and guidance in analyzing our data. Our thanks also go to Kurosh Keikanzadeh and Kim Nguyen; both have helped us tremendously with the laboratory aspect of this research. Our last and greatest thanks goes to Dr. Petra Wilder-Smith. We are very thankful for her continual assistance and mentorship throughout this project and the writing of this article. Without all of their help, we would not have been able to complete this project and article.
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Footnotes
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Ms. Sarkissian and Ms. Le are both undergraduate students at the University of California, Irvine, expected to graduate in 2005. Direct correspondence and requests for reprints to Ms. Ani Sarkissian, 123 Adams St., Glendale, CA 91205; 818-281-7956 phone; anisarkiss{at}aol.com.
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Abstract
Materials and Methods
