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ORIGINAL RESEARCH Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 3  |  Page : 261-267
In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study


1 Department of Oral Medicine and Radiology, Government Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Medical Physics, Anna University, Chennai, Tamil Nadu, India

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Date of Submission17-Jun-2008
Date of Decision02-Feb-2009
Date of Acceptance03-Mar-2009
Date of Web Publication30-Oct-2009
 

   Abstract 

Aims and Objectives: To compare the autofluorescence spectra of oral submucous fibrosis (OSF) with normal mucosa, the autofluorescence spectra of OSF before and after treatment with intralesional dexamethasone and hyaluronidase, the clinical improvement following treatment with the changes in autofluorescence spectra and to prove that autofluorescence spectroscopy is a good method for diagnosis and assessment of treatment effectiveness in OSF.
Materials and Methods: The study was conducted at the Department of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospital, Chennai and Division of Medical Physics and Lasers, Department of Physics, Anna University, Chennai in 20 patients seeking medical management for symptomatic OSF and 20 patients who had dental caries only without any oral mucosal diseases and oral habits were used as normal controls. Their ages ranged from 20 to 40 years, including both male and female. In vivo fluorescence emission spectra were obtained using a handheld optical fiber probe attached to a Fluoromax-2 spectrofluorometer.
Results: The fluorescence spectrum of OSF had an intense fluorescence emission at 385 nm with a secondary emission peak at 440 nm compared with that of the normal oral mucosa. The average fluorescence spectrum of the post treated OSF mucosa had a lesser intensity around 385 nm and a higher intensity around 440 nm than that of the pre treated OSF mucosa, thereby mimicking the normal oral mucosa. All the three clinical parameters (maximal mouth opening, tongue protrusion and the severity of burning sensation) showed a high statistical significance, with P < 0.001, as in the case of classification of pre treated OSF mucosa from the post treated OSF mucosa using the autofluorescence technique.
Conclusion: The change in the fluorescence emission spectrum for both normal and OSF mucosa before and after treatment can be explained by analyzing the changes in the fluorescence intensity of the endogenous fluorophores.

Keywords: Autofluorescence spectroscopy, dexamethasone, hyaluronidase, OSF

How to cite this article:
Vedeswari C P, Jayachandran S, Ganesan S. In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study. Indian J Dent Res 2009;20:261-7

How to cite this URL:
Vedeswari C P, Jayachandran S, Ganesan S. In vivo autofluorescence characteristics of pre- and post-treated oral submucous fibrosis: A pilot study. Indian J Dent Res [serial online] 2009 [cited 2017 Sep 20];20:261-7. Available from: http://www.ijdr.in/text.asp?2009/20/3/261/57354
When biologic tissue interacts with light, it becomes excited and reemits light of varying colors (fluorescence) [1],[2] , which can be detected by sensitive spectrometers. Compared with normal tissues, different diseased tissues contain different morphohistologic characteristics and intrinsic fluorophores that give rise to different fluorescence emission spectra when the tissues are excited at a suitable wavelength. [1],[3] The physical and chemical properties of a tissue can be evaluated by analyzing the intensity and the character of light emitted in the form of fluorescence. [9],[10] Oral submucous fibrosis (OSF) is characterized by increased collagen deposition in the subepithelial connective tissue layer and markedly atrophic oral epithelium. It has been demonstrated that, under the range of a 300-360 nm excitation wavelength, the emission band at 380-400 nm is mainly attributed to the presence of collagen, whereas that at 440-460 nm is mainly due to the presence of nicotinamide adenine dinucleotide (NADH). [6],[7] Conventional submucosal injections of dexamethasone plus hyaluronidase results in symptomatic improvement in 83% of the patients. [8] Steroids act by decreasing inflammation, decrease in fibroblastic proliferation and deposition of collagen. Hyaluronidase acts by break-down of the hyaluronic acid matrix, lowering the viscosity of the intercellular cementing substance and also decreasing collagen formation. This study was conducted to evaluate the possibility of using endogenous fluorophores present in the OSF mucosal sites to diagnose and to monitor the therapeutic response by analyzing their native fluorescence characteristics. [6],[12],[13],[14]

The aims and objectives of this study were

  • To compare the autofluorescence spectra of the OSF with the normal mucosa.
  • To compare the autofluorescence spectra of the OSF before and after treatment with intralesional dexamethasone and hyaluronidase.
  • To compare the clinical improvement following treatment with the changes in the autofluorescence spectra.
  • To prove that autofluorescence spectroscopy is a good method for diagnosis and assessment of treatment effectiveness in OSF.



   Materials and Methods Top


The study was conducted at the Department of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospital, Chennai, and Division of Medical Physics and Lasers, Department of Physics, Anna University, Chennai, after obtaining approval. The study population included 20 patients who were seeking medical management for symptomatic OSF and 20 patients who had dental caries only without any oral mucosal diseases and oral habits were used as normal controls. Their ages ranged from 20 to 40 years, including both male and female. A positive history of chewing of the areca nut or one of its commercial preparations, difficulty in swallowing and chewing and burning sensation on eating spicy foods and restricted mouth opening and changes in the oral mucous membrane, including the presence of palpable fibrotic bands, stiffness and blanching, were used to establish the diagnosis. Some OSF patients also had depapillation of the tongue and impaired tongue mobility. History of habits, including duration in months and frequency per day, duration of symptoms, oral mucosal sites of involvement, the maximal mouth opening, tongue protrusion and the severity of burning sensation were recorded in a structured proforma designed for this study. The severity of burning sensation was graded from 0 to 3 as no burning sensation, mild, moderate and severe. Mouth opening was measured as the interincisal distance from the mesioincisal edge of the upper left central incisor tooth to the mesioincisal edge of the lower left central incisor tooth. Measurement was made using a geometric divider and scale and was recorded in millimeters. The degree of tongue protrusion was recorded in units of 5 mm from the incisal edge of the lower teeth by viewing the protruded tongue from the lateral aspect of the head and measuring the distance from the mesial contact area of the lower central incisors to the tip of the protruded tongue. When the value was found to lie between units of 5 mm, it was adjusted to the higher limit. This was carried out to allow for the fine movement of the tip that did not permit an exact millimeter recording. The patients were graded clinically as follows: Grade I, only blanching of the oral mucosa without symptoms; grade II, grade I and burning sensation, dryness of the mouth, vesicles or ulcers in the mouth without tongue involvement; grade III, in addition to grade II, restriction of mouth opening; grade IV, in addition to grade III, palpable bands all over the mouth without tongue involvement; grade V, grade IV and tongue also involved; grade VI, OSF along with histopathologically proven oral cancer. Patients with systemic diseases like diabetes, hypertension, hepatic or renal diseases, pregnant and lactating mothers and any kind of allergy were excluded from the study. All the lesions were diagnosed based on history and clinical examination. Basic blood and urine investigations were carried out for all the patients.

Autofluorescence spectroscopy

Reassurance and counseling was given and informed consent was obtained from the patient. In vivo fluorescence emission spectra were obtained from each OSF buccal mucosal site and each normal buccal mucosal site of the control patients using a handheld optical fiber probe attached to a Fluoromax-2 spectrofluorometer (ISA Jobin Yvon - Spex, Edison, New Jersey, USA). A monochromator with a 150-W ozone-free Xenon lamp provided the excitation light [Figure 1] and [Figure 2]. The desired excitation wavelength and the emission spectrum were selected by a PC-controlled monochromator. The excitation light was guided to illuminate samples by one arm of a Y-type quartz fiber bundle and the emission fluorescence was collected by another arm of the fiber bundle and directed to the photomultiplier detector. The signal was then amplified and displayed on the computer monitor. All Fluoromax-2 functions are controlled by the DataMax software (Dover corporation (NYSE:DOV), Orlando, Florida, USA), which communicates between a PC- compatible computer and the Fluromax-2. The optical fiber probe was disinfected with 2.4% gluteraldehyde solution, rinsed with phosphate-buffered saline and wrapped with a transparent plastic membrane before each clinical use.

Excitation-emission spectra

In this study, ultraviolet light at 330 nm wavelength was used to excite each sample and the resulting emission spectra were recorded from 350 to 600 nm in 1 nm increments. During measurement, the probe was gently touched on the surface of the normal or lesional buccal mucosal site. The spectrum at each site was obtained by averaging three spectra measured from three adjacent sites (2 mm apart) of the same lesion.

Treatment protocol

Then, patients of the study group were subjected to a treatment protocol of intralesional injection of a combination of dexamethasone sodium phosphate 4 mg/ml and hyaluronidase 1500 IU twice a week for 6 weeks. They were also given antioxidants and micronutrients supplementation and were taught muscle-stretching exercises. At each visit, following topical application of lignocaine 2%, 1500 IU of hyaluronidase was dissolved in 2.0 ml of dexamethasone sodium phosphate in a 2 ml disposable syringe. The drugs were injected at multiple sites submucosally by means of a gauge 24 needle, not more than 0.2 ml solution per site. The patient was evaluated at each visit during the treatment period of 6 weeks. A clinical examination was carried out at every visit and the parameters of maximal mouth opening, tongue protrusion and burning sensation were recorded.

Emission spectra after treatment

After 6 weeks, the fluorescence emission spectra of the treated site were recorded as before. There were no adverse effects throughout the study.

Statistical analysis

After normalization, the mean (± SD) fluorescence intensities at 385± 5 nm (I 385± 5 nm ) and 440± 5 nm (I 440± 5 nm ) emission peaks and the mean ratio of I 440± 5 nm /I 385± 5 nm were calculated for the normal oral mucosa and OSF mucosa before and after treatment. The differences in the mean values of I 385± 5 nm , I 440± 5 nm and ratio of I 440± 5 nm /I 385± 5 nm between the normal oral mucosa and the pre treatment OSF mucosa and between the normal oral mucosa and the post treatment OSF were assessed for statistical significance by unpaired Student's t- test. The differences in the mean values of I 385± 5 nm , I 440± 5 nm and ratio of I 440± 5 nm /I 385± 5 nm between the pre treatment OSF and the post treatment OSF were assessed for statistical significance by paired Student's t-test. The mean (± SD) values of maximal mouth opening, tongue protrusion and burning sensation of the pre treatment OSF group were compared with that of the post treatment OSF group using paired Student's t-test. A P-value of less than 0.05 was considered to be statistically significant.


   Results Top


Comparison of autofluorescence characteristics of the normal oral mucosa and the OSF mucosa

All the spectra were normalized by dividing the intensity of each wavelength by the maximum intensity of the emission spectrum to avoid the intensity variations due to different excitation light power, fluorescence collection efficiency as well as interpatient variations. The normalized fluorescence spectra of the normal oral mucosa and the OSF mucosa are shown in Figure 3. The fluorescence spectrum of OSF had an intense fluorescence emission at 385 nm, with a secondary emission peak at 440 nm, compared with that of the normal oral mucosa. In order to find out whether any considerable statistical significance exists in the spectral signature between normal oral mucosa and OSF mucosa, three different ratio parameters, such as R1 = I 440± 5 nm /I 385± 5 nm [Table 1] , R2 = I 420± 5 nm /I 370± 5 nm and R3 = I 470± 5 nm /I 450± 5 nm were introduced. They were subjected to Unpaired Student's t-test. In this context, the ratio value R1 is considered for further analysis and discussion. When the mean (± SD) fluorescence intensities at 385± 5 nm (I 385± 5 nm) and 440± 5 nm (I 440± 5 nm ) emission peaks and the mean ratio of I 440± 5 nm /I 385± 5 nm were compared between the groups, the OSF group had a significantly higher mean value of I 385± 5 nm , a significantly lower mean value of I 440± 5 nm and a significantly lower mean ratio of I 440± 5 nm /I 385± 5 nm than the normal oral mucosa (P < 0.001 ** ) [Table 1]. The scattered graph is plotted between the intensity ratios of I 440± 5 nm /I 385± 5 nm and the number of subjects. From the scatter plot, it was observed that there is a distinct variation in the distribution of R1 value between the normal oral mucosa and the OSF mucosa [Figure 4]. It is found that if the ratio value R1 is greater than 0.96, the tissue is normal oral mucosa, whereas at values lesser than 0.96, the tissue is OSF mucosa. Based on the R-value 0.96, the diseased persons are classified with 90% sensitivity and normal oral mucosa with 80% specificity.

Comparison of normal oral mucosa and pre treated and post treated OSF mucosa

In this context, the post treated OSF cases were subjected to measurement of fluorescence at identical conditions, i.e. as in the case of measuring the pre treated OSF condition. The post treated OSF mucosa had a lower mean value of I 385± 5 nm and I 440± 5 nm and a higher mean ratio of I 440± 5 nm /I 385± 5 nm compared with that of the normal oral mucosa [Table 2],[Figure 5]. The fluorescence emission spectra of the pre- and post treated OSF groups are shown in Figure 6. The average fluorescence spectrum of the post treated OSF mucosa had a lesser intensity at around 385 nm and a higher intensity at around 440 nm than that of the pre treated OSF mucosa, thereby mimicking the normal oral mucosa [Table 3]. Scatter plot drawn for I 440± 5 nm /I 385± 5 nm for the pre- and post treated cases showed R factor of 0.96 to separated the pre treated from the post treated cases [Figure 7]. If R-value is < 0.96, the tissues are pre treated OSF mucosa and for R-value > 0.96, the tissues are post treated OSF mucosa. Thus, it is interestingly observed that the ratio values of the post treated OSF group have enhanced to greater than 0.96, as in the case of normal subjects, indicating the response of treatment and the transformation of the OSF toward normalcy.

Comparison of treatment response between autofluorescence and conventional clinical methods

In order to verify whether the autofluorescence technique is comparable with that of conventional clinical methods to monitor the treatment response, the pre- and post OSF patients were subjected to measurement of maximal mouth opening, tongue protrusion and the severity of burning sensation [Figure 8]. The mean± SD of these three parameters was also estimated [Table 4].

The mean (± SD) maximal mouth opening of the post treated OSF group was significantly higher than that of the pre treated OSF group (P < 0.001 ** ). The mean (± SD) tongue protrusion of the post treated OSF patients was significantly higher than that of the pre treated oral submucous fibrosis patients (P < 0.001 ** ) [Figure 6]. The mean (± SD) severity of burning sensation of the post treated OSF patients was significantly lower than that of the pre treated OSF patients (P < 0.001 ** ). All the three clinical parameters showed high statistical significance, with P < 0.001, as in the case of classification of the pre treated oral submucous fibrosis mucosa from the post treated OSF mucosa, using the autofluorescence technique. Further, from the scattered plot, it is verified that the ratio value 0.96 is greater for both the normal oral mucosa and the post treated OSF mucosa. Of the 20 treated patients, only one patient's R-value was < 0.96, indicating that autofluorescence may also be effectively used to monitor the therapeutic response.


   Discussion Top


In this study, we found that with ultraviolet light at 330 nm excitation, the spectra of the OSF mucosa had an intense fluorescence emission at 385 nm and a secondary emission peak at 440 nm with that of the normal oral mucosa. In addition, the pre treated OSF mucosa had a significantly higher mean value of I 385 ± 5 nm , a significantly lower mean value of I 440 ± 5 nm and a significantly lower mean ratio of I 440 ± 5 nm /I 385 ± 5 nm than the normal oral mucosa (P < 0.001). Previous studies have shown that at 330 nm excitation, the 380 and 460 nm emission peaks of the autofluorescence spectra for oral mucosal tissues reflect the collagen content in the subepithelial connective tissue and the NADH content in the epithelial cells, respectively. [7],[10],[12] Therefore, the increased subepithelial collagen content could explain why the pre treated OSF mucosa had a significantly higher intensity of the 385 nm emission peak of the autofluorescence spectra than the normal oral mucosa and the post treated OSF mucosa. Furthermore, compared with the normal oral epithelium, the markedly atrophic OSF epithelium allowed more excitation energy to penetrate into the subepithelial connective tissue and more collagen- derived emission fluorescence to go through. This factor also contributed to the high intensity of the 385 nm emission peak of the autofluorescence spectra of the OSF mucosa. In addition, the markedly atrophic OSF epithelium might contain less amount of NADH than the normal oral epithelium. The fibrosis-induced reduction of blood vessels in the lamina propria might also diminish the metabolic rate of the oral epithelial cells, which, in turn, results in a lower NADH content in the OSF epithelium. Therefore, the OSF mucosa had a significantly lower intensity of the 440 nm emission peak than the normal oral mucosal sites. This suggests that autofluorescence spectroscopy can detect the increased amount of collagen in the subepithelial connective tissue and the decreased amount of NADH in the epithelium. The monochromatic light signal penetrates only about 500 µm deep into the tissue. Thus, this technique is capable of analyzing only the most superficial portions of the oral lesions. [9] In this study, no significant differences in the pattern of the 385 and 440 nm emission band was found between the normal oral mucosa and the post treated OSF mucosa.

Steroids are well known to act as antiinflammatory agents for prevention or suppression of the fibroproductive inflammation found in the OSF mucosa thus ameliorating this fibrocollagenous condition. [5] Tsai et al. had suggested that in addition to the known mechanisms, a corticosteroid-induced increase in collagen degradation could result from enhanced collagen phagocytosis by the fibroblast. [11] Hyaluarnidase degrades the hyaluaronic acid matrix actively, promoting lysis of the fibrinous coagulum. [4] It is due to the above actions that the collagen content of the OSF mucosa had reduced and the atrophic oral epithelium had healed. Hence, following the treatment, there is a similar fluorescence characteristic as in the case of the normal oral mucosa. The improvement in mouth opening, tongue protrusion and decrease in the burning sensation when compared with the initial stage was highly significant and correlated well with the autofluorescence characteristics. This again is attributed to treatment efficacy, resulting in collagen degradation and decrease in the inflammation.


   Conclusion Top


In summary, the change in the fluorescence emission spectrum for both the normal and the OSF mucosa before and after treatment can be explained by analyzing the changes in the fluorescence intensity of endogenous fluorophores. This study adds evidence that high collagen and low NADH characterize the OSF mucosa. It is also obvious from this study that autofluorescence spectroscopy can monitor the therapeutic response and characterize the tissue transformation following treatment. Furthermore, investigations with a large group of experimental subjects will also be useful for the development of a statistical database and a user-friendly diagnostic algorithm that could facilitate early detection. Autofluorescence spectroscopy being a non invasive and easily applicable tool for the detection of the alterations in the structural and biochemical composition of cells may reduce the patient morbidity and, therefore, is of great clinical importance. From this study, we conclude that autofluorescence spectroscopy provides valuable information for the diagnosis and also for monitoring the therapeutic response in OSF. However, results of this study on autofluorescence characteristics in the diagnosis and therapeutic monitoring in OSF has to be validated with more studies involving large samples and longer follow-up.


   Acknowledgments Top


We express our sincere thanks to our Principal, the faculties in the Departments of Oral Medicine and Radiology, Tamilnadu Government Dental College and Hospital, and Mr. Sivabalan, Research Scholar, Department of Medical Physics, Anna University, for their valuable suggestions, kind help and encouragement in the present study.

 
   References Top

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2.Izatt RM, Christensen JJ. Heats of proton ionization, pK and related thermodynamic quantities. In: Fasman GD, editor. Handbook of biochemistry and molecular biology. 3 rd ed. Cleveland: CRC Press; 1975;205-10.   Back to cited text no. 2      
3.Fasman GD (Ed.). Handbook of biochemistry and molecular biology. 3 rd ed. Cleveland: CRC press; 1975; 205-10.  Back to cited text no. 3      
4.Kakar PK, Puri RK, Venkatachalam VP. Oral submucous fibrosis - treatment with hyalase. J Laryngol Otol 1985;99:57-9.  Back to cited text no. 4      
5.Gupta D, Sharma SC. OSF - A new treatment regimen. J Oral Maxillofac Surg. 1988;46:830-3.  Back to cited text no. 5      
6.Glasgold R, Glasgold M, Savage H, Pinto J, Alfano R, Schantz S. Tissue auto-fluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis. Cancer Lett 1994;82:33-41.  Back to cited text no. 6      
7.Bottiroli G, Croce AC, Locatelli D, Marchesini R, Pignoli E, Tomatis S, et al. Natural fluorescence of normal and neoplastic human colon: A comprehensive 'ex-vivo' study. Lasers Surg Med 1995;16:48-60.  Back to cited text no. 7      
8.Lai DR, Chen HR, Lin LM, Huang YL, Tsai CC. Clinical evaluation of different treatment methods for OSF. A 10-year experience with 150 cases. J Oral Pathol Med 1995;24:402-6.  Back to cited text no. 8      
9.Gillenwater A, Jacob R, Richards-Kortum R. Fluorescence spectroscopy: A technique with potential to improve the early detection of aero digestive tract neoplasia. Head and Neck 1998; 20:556-62.  Back to cited text no. 9      
10.Chen CT, Chiang HK, Chow SN, Wang CY, Lee YS, Tsai JC, et al. Auto- fluorescence in normal and malignant human oral tissues and in DMBA-induced hamster buccal pouch carcinogenesis. J Oral Pathol Med 1998;27:470-4.  Back to cited text no. 10      
11.Tsai CC, Ma RH, Shieh TY. Deficiency in collagen and fibronectin phagocytosis by human buccal mucosa fibroblasts in vitro as a possible mechanism for OSF. J Oral Pathol Med 1999;28:59- 63.  Back to cited text no. 11      
12.Chen HM, Wang CY, Chen CT, Yang H, Kuo YS, Lan WH, et al. Auto- fluorescence spectra of OSF. J Oral Pathol Med 2003;32:337-43.  Back to cited text no. 12      
13.Tsai T, Chen HM, Wang CY, Tsai JC, Chen CT, Chiang CP. In vivo auto- fluorescence spectroscopy of oral premalignant and malignant lesions. Distortion of fluorescence intensity by submucous fibrosis. Lasers Surg Med 2003;33:40-7.  Back to cited text no. 13      
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Correspondence Address:
S Jayachandran
Department of Oral Medicine and Radiology, Government Dental College and Hospital, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.57354

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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