|Year : 2015 | Volume
| Issue : 4 | Page : 161-166
Expression of type I and type III collagens in oral submucous fibrosis: An immunohistochemical study
Venkatesh V Kamath, Komali Rajkumar, Abhay Kumar
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College Hospital and Research Centre, Bengaluru, Karnataka, India
|Date of Web Publication||17-Feb-2016|
Venkatesh V Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College Hospital and Research Centre, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Oral submucous fibrosis (OSMF) is a potentially malignant collagen - metabolic disorder linked to consumption of betel quid and areca nut. The deposition of collagen and its major subtypes have been the subject of intense scrutiny in the etiopathogenesis of the disorder. Aims and Objectives: The present study was planned to immunohistochemically identify the expression of collagens I and III (COL I and III) in different grades of OSF and compare it with normal oral mucosa and scar tissue. Materials and Methods: Archival paraffin sections of 72 cases of various grades of OSMF, ten cases of normal mucosa as controls and four cases of scar tissue were stained with antibodies to COL I and III (BioGenex Laboratories, CA, USA) to evaluate the collagen subtypes on paraffin sections. The expression was quantified by image analysis software (Jenoptik Optical System, ProgRes ® Capture Pro, version 2.8.8) and statistically analyzed. Results: COL I and III stained all the tissues ubiquitously. COL I was more in ratio and quantity in all the grades of OSMF, normal mucosa, and scar tissue. The proportion of COL I to COL III seemed to increase with progressive grades of OSF. Interestingly, during the process of fibrosis COL III seems to be deposited earlier and gradually replaced by COL I resulting in a skewed ratio vis a vis normal oral mucosa and scar tissue. Conclusions: COL I expression was found to be proportionate with advancing grades of OSF while COL III expression increased in Grade I but subsequently decreased as severity of OSF increased. The increase in COL I at the expense of COL III showed a similar pattern in the submucosa while in the deeper muscle only Grade III cases reflected the trend. While all cases of OSF revealed excessive expression in comparison with normal oral mucosa, the comparison with scar tissue was variable.
Keywords: Collagen I, collagen III, immunohistochemistry, oral submucous fibrosis
|How to cite this article:|
Kamath VV, Rajkumar K, Kumar A. Expression of type I and type III collagens in oral submucous fibrosis: An immunohistochemical study. J Dent Res Rev 2015;2:161-6
|How to cite this URL:|
Kamath VV, Rajkumar K, Kumar A. Expression of type I and type III collagens in oral submucous fibrosis: An immunohistochemical study. J Dent Res Rev [serial online] 2015 [cited 2019 Jul 16];2:161-6. Available from: http://www.jdrr.org/text.asp?2015/2/4/161/176680
| Introduction|| |
The deposition of collagen in OSF has always been a major area of scrutiny in the understanding of the pathogenesis of the disorder. OSF is a potentially malignant oral disorder that is, unique in its presentation and geographical predisposition. Almost always associated with the habit of areca nut chewing the proliferation of commercial preparations of the nut has propelled this disorder into endemic proportions. Recent reports indicate about 5 million people perusing the nut in its various forms in almost all parts of the world. 
The fibrosis in the submucosa is pathognomic of the condition and the collagen, and extracellular matrix (ECM) component content of the tissue has been scrutinized constantly. Collagen is a group of naturally occurring protein family, forming one of three essential proteins of the skin (keratin and elastin being others). Collagens are most abundant (almost 30% of the total amount of proteins), insoluble, extracellular, glycoprotein's in the human body, thus forming an essential structural component of all connective tissues and are also found in the interstitial tissues of all parenchymal organs. They contribute to the stability of the tissues and organs and maintain their structural integrity in the body. 
Collagen framework for lamina propriae and submucosa is produced by the fibroblasts present in the oral mucosa. Distribution of collagen in adult oral mucosa is: 80-90% comprised by collagen type I (COL I), 10-15% formed by collagen type III (COL III) and others represents <5% of total. However, in the fetal oral mucosa, COL III is predominant, which is suggestive of developmental alteration in the synthesis and degradation of the relative collagen types, as the mucosal structure matures. 
In oral mucosa, collagen fibers seen are of indeterminate length, and their diameter varies from 1 to 12 μm. These fibers are loosely or densely packed but have straight or slightly wavy course. 
COL I is long, thin fibril and found in profusion in most connective tissues, having enormous tensile strength. It is a heterotrimer, consisting of two identical alpha 1 (I) chains and one alpha 2 (I) chain.  COL I alpha I is a human gene that encodes the major component of COL I and is located on chromosome 17. But COL III is a homotrimer, consisting of three identical alpha 1 (III) chains and encoded by COL III alpha 1 gene located on chromosomes 2. COL III is frequently present in association with COL I as COL III is widely distributed in COL I containing tissues with the exception of bone.  Thus, this homotrimeric molecule often contributes to mixed fibrils with COL I. 
Studies on the possible causes and modulations of the excessive fibrosis in OSF have yielded confusing results. Though it is generally accepted that the pathway of deposition of the excess fibers follows the ones seen during healing of oral wounds the nature of the collagen suggests a probable reorientation and replacement of the normal collagen. The chewing of areca nut releases its active alkaloids arecoline and arecaidine that are established fibrogenetic factors. The accessory constituents of the nut, the polyphenols especially tannins, and flavonoids are known collagen stabilizing agents preventing collagenase-mediated remodeling. The net effect, hypothetically, results in over-deposition of a resistant form of collagen reflected in the tissue as fibrosis. ,,
This study aims to identify and quantify the collagen types in OSF using monoclonal COL I and COL III antibodies and compare the findings with those of normal oral mucosa and scar tissues.
| Materials and Methods|| |
Seventy-two cases of OSF, ten cases of normal buccal mucosa and four cases of skin scar tissue were evaluated immunohistochemically (IHC) for COL I and COL III expression. Histopathological grading of OSF was done on hematoxylin and eosin stained sections based on Pindborg and Sirsat classification criteria.  Paraffin-embedded formalin-fixed tissues cut at 4-micron thickness were used for IHC evaluation. Primary antibodies for COL I and COL III and polymer horseradish peroxidase detection system (BioGenex Laboratories, CA, USA) were used for the IHC procedure as directed by the manufacturer. Microwave antigen retrieval method was performed using tris-EDTA buffer. Immunolabeling was done at 4°C and tissues were incubated overnight with COL I antibody whereas for COL III antibody, incubation was done for 45 min.
Positive cases were graded according to their intensity as mild (Score 1), moderate (Score 2) and intense (Score 3).
Quantitative analysis of the collagens expression was done by image analysis software (Jenoptik Optical System, USA, ProgRes ® Capture Pro, version 2.8.8). The data obtained was tabulated and subjected to statistical analysis (SPSS, version 18.0) (IBM Systems Inc, USA).
| Results|| |
The overall trend revealed moderate staining (Score 2) for COL I in normal buccal mucosa and in Grades I and II cases of OSF [Figure 1], [Figure 2] and [Figure 3]. Intense staining (Score 3) for COL I was seen in scar tissue [Figure 4] and in Grade III cases of OSF. Tissues stained moderately (Score 2) for COL III in all the study cases [Table 1].
|Figure 1: Photomicrograph of a Grade I OSF case showing expression of collagen I antibody expression. Note the depth and intensity of the expression (×10)|
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|Figure 2: Photomicrograph of a Grade I OSF case showing expression of collagen III antibody expression. Note the decreased intensity and decreased footprint of the antibody expression (×10)|
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|Figure 3: Photomicrograph of a Grade III OSF case showing expression of collagen I antibody expression. Note the increased intensity especially in the sub-epithelial areas where hyalinization and density of collagen are usually seen (×10)|
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|Figure 4: Photomicrograph of a case of scar tissue showing expression of collagen I antibody expression. Note the diffuse and almost total expression of the antibody in the sub-epithelial connective tissue (×10)|
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The quantitative expression of COL I and COL III between different study groups was analyzed by one-way ANOVA test.
Comparison of quantitative expression of collagen I and III in submucosa in varying grades of OSF
COL I expression was found to be more compared to that of COL III in all the three grades of OSF and the mean difference was found to be statistically significant [Table 2] and [Graph 2].
|Table 2: Quantitative expression of collagens I and III in submucosa (one-way ANOVA test)|
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Comparison of quantitative expression of collagen I and III in muscle in varying grades of OSF
COL III expression was found to be more compared to COL I in Grade I OSF, however, the mean difference was not found to be statistically significant. In Grade II OSF, COL I expression was found to be more, but their mean difference was not statistically significant. COL I expression in Grade III OSF was found to more than COL III and the mean difference was found to be statistically significant [Table 3] and [Graph 3].
|Table 3: Quantitative expression of collagens I and III in muscle (one-way ANOVA test)*|
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Comparison of quantitative expression of collagens I and III in totality amongst various grades of OSF
The expression of COL I progressively increased with increasing grades of OSF. However, very little change was evident in the expression of COL III. This was borne out by the lack of statistical significance of the results [Table 4] and [Graph 1].
|Table 4: Proportion of total collagens I and III expression in the various grades of OSF*|
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Ratio of collagen I: III in various groups
The replacement of COL III with COL I was reflected in the increasing ratios of the latter in almost all grades of OSF and in scar tissue. Interestingly, the ratio was found to be the highest in normal mucosa and scar tissue in the submucosal region. In the OSF tissues there seemed to be a trend of initially increased deposition of COL III which was subsequently replaced by COL I [Table 5] and [Graph 4].
| Discussion|| |
The over-deposition of collagen fibers is pathognomic in the condition of oral submucous fibrosis (OSMF). This is directly linked to the clinical presentation of the pale oral mucosa, fibrotic bands and trismus and probably the potential for malignant transformation. 
Studies on the type and pathogenesis of the fibrotic processes have been the focus of researchers working on the condition. One of the initial attempts has been by Binnie and Cawson  who examined the type of collagen in a 32-year-old patient with OSF. They found that the collagenous fibrils are thinner than normal connective tissue, are of the embryonic type and that the defect may lie in polarization and maturation. They hypothesized that there was the excess production of collagen fibrils showing increased argyrophilia. In another study, in early stages of OSMF, COL III, tenascin, fibronectin were found to be characteristically enhanced in the lamina propria and the submucosal layer. In the advanced stage, COL III, fibronectin, tenascin depositions decreased and were entirely replaced with COL I only. The result indicates that the ECM remodeling steps in OSF are similar to each phase of usual granulation tissue formation. Restricted mouth opening may be a result of the loss of variety of ECM molecules including elastin in the homogeneity of COL I replacing muscle fibers. 
In an electron microscopic study on OSMF, it was noted that collagen was packed in dense bundles in the lamina propria, reaching close to the epithelial-connective tissue junction, and deeper to the blood vessels and muscle fibers. Immunofluorescent microscopy and special staining with Sirius red and polarization microscopy demonstrated that COL I fibrils which are larger than 40 nm in diameter, form the bulk of collagen and type III, which varies from 5 to 40 nm in diameter, is localized in the above-mentioned sites. It was also concluded that there is no reason on a morphological basis, to believe that collagen of submucous fibrosis is abnormal. 
In this study, the expression of the COL I and III antibodies was ubiquitous in all tissues, albeit with varying intensity and localization. In early grades of OSF, a parallel increase in the expression of both the subtypes was noted. While the expression of COL I followed a curve proportionate with increasing grades of OSF peaking at Grade III and in scar tissue, COL III expression showed a drop in Grades II and III of OSF. This probably indicates a replacement of COL III by COL I. The present finding is generally in concordance with previous studies. The use of monoclonal antibodies obviates the possibility of cross-reactivity of the collagen molecules, and thus the tissue expression mirrors the replacement of COL III by COL I. The latter is more stable, resistant to the effects of degradatory enzymes involved in tissue remodeling and is thus reflective of the sustained fibrosis seen in the disorder.
The demographic mapping of the collagen obtained from quantification by image analysis revealed the proportion of collagen in the sub-epithelial regions. The predominance of COL I was seen over COL III in a ratio of 1.1-1.3. Interestingly, there seemed to be a decrease of the ratio in all grades of OSF as compared with normal oral mucosa and scar tissue, especially in the submucosal region.
COL I is more mature and stable due to crosslinking as compared with COL III. As the collagen in all tissues in a state of dynamic flux, the remodeling processes probably account for the variability of the ratios seen in our study. Similar results have been expressed in previous studies where COL III enhancement have been reported.  Fibroblast culture studies in OSF have revealed the predominance of F3 fibroblasts, known to deposit stable COL I, indicating the subtle shift in cellular mechanisms of fibrosis. 
In vitro studies on human fibroblasts using areca extracts or chemically purified arecoline support the theory of fibroblastic proliferation and increased collagen formation that is, also demonstrable histologically in human OSF tissues. Hydrolysis of arecoline produces arecaidine that has pronounced effects on fibroblasts. There was a concentration-dependent stimulation of collagen synthesis when fibroblasts were exposed to both arecoline and arecaidine. However, the stimulation was always greater with arecaidine. This suggests that arecaidine is active metabolite in fibroblast stimulation. This view was further supported by the finding that, the addition of slaked lime to arecanut in pan, facilitates hydrolysis of arecoline to arecaidine making this agent available in the oral environment. 
In a study by Kuo et al.,  fibroblasts derived from OSF specimens were isolated and established in cultures and were compared with the normal buccal mucosa fibroblasts in terms of collagen biosynthesis. When the relative amounts of collagen synthesis were estimated using gel electrophoresis and slab gel fluorography, it was found that the OSF and normal cells produced about 85% COL I and 15% COL III. The ratio of the alpha 1 (I) to alpha 2 (I) chain was 3:1 in OSF compared to 2:1 expected for COL I. It was also found that there was higher than normal values of procollagen mRNA. On comparing a gene copy number of pro-alpha 2 (I) collagen, it was found to be 1.05 in respect to 1.0 found in normal. There was an increased 1.5-fold of collagen biosynthesis in the formation of collagen in OSF. 
In a comparative study on type of collagens in scar and keloid tissue, it was found that that the ratio of COL I to COL III has certain differences in the magnitude and expression of collagen genes and posttranslational collagen modifications in the tissue of hypertrophic and keloid scars. The authors also suggested age-specific time course of collagen metabolic and structural changes in the abnormal scars may result from their alterations of the population composition of fibroblasts with a different phenotype.  The intensity and type of collagen deposition in these tissues has been attributed to gene regulation thereby establishing an intrinsic genetic pathway of the fibrosis. 
The pattern of collagen deposition seems to be rather complex in the disorder of OSF. Increased deposition of COL I and COL III with a subsequent shift in the ratio towards the more stable COL I subtype seems to be the order. Various factors including tissue remodeling, fibroblast phenotype, fibrotic pathways, and the actions of areca nut constituents seem to be at play in this puzzle. The net result is undoubtedly increased fibrosis that afflicts the tissue dynamics reflected in the clinical condition.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]