|Year : 2014 | Volume
| Issue : 2 | Page : 57-64
The nature of collagen in oral submucous fibrosis: A systematic review of the literature
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Bangalore, Karnataka, India
|Date of Web Publication||12-Aug-2014|
Dr. V V Kamath
Department of Oral and Maxillofacial Pathology, Dr. Syamala Reddy Dental College, Hospital and Research Centre, Munnekolala, Marathalli, Bangalore - 560 037, Karnataka
Source of Support: None, Conflict of Interest: None
Background: The nature of collagen in the oral potentially malignant disorder of oral submucous fibrosis (OSF) has always been a subject of interest to researchers studying the condition. The type, constitution, and morphology of the collagen has been studied to understand the pathogenesis of the disorder.
Aim: The present review attempts to collate literature to assess the changes reported on the nature of collagen in OSF.
Materials and Methods: A systematic analysis of literature based on the PRISMA protocol was carried out on studies that analyzed the nature of collagen in OSF. A total of 30 articles were found acceptable for analysis. The reported literature was categorized into histological, immuno-histochemistry (IHC), electron microscopic (EM)/scanning electron microscopic (SEM), biochemical, culture, and genetic studies.
Results: Light microscopic studies revealed increased collagen with compaction and condensation as the disease advanced. Polarising microscopy revealed presence of greenish yellow (type I) thick fibers replacing the yellowish orange (type III) thin fibers in the submucosa. Fibrosis originated in the submucosa and subsequently involved the lamina propria as the disease advanced. EM/SEM investigations revealed frayed ends, degeneration of collagen cores. Increased expression of collagen antibodies and total collagen content were observed in IHC and biochemical studies, respectively. Cultured fibroblasts from OSF tend to produce more collagen in comparison to normal fibroblasts indicating a phenotypic change. Increased expression and upregulation of collagen genes were consistent in OSF cases.
Conclusion: The collagen deposition in OSF is biochemically normal but quantitatively more. Fibroblast phenotypic change, upregulation of collagen genes, and selective remodeling due to decreased degradation seem to contribute to the increased fibrosis in the disorder.
Keywords: Collagen I, collagen III, oral submucous fibrosis
|How to cite this article:|
Kamath V V. The nature of collagen in oral submucous fibrosis: A systematic review of the literature. Saudi J Oral Sci 2014;1:57-64
| Introduction|| |
The increased deposition of collagen (fibrosis) in the potentially malignant oral disorder of oral submucous fibrosis (OSF) is pathognomic of the condition. Ever since its categorization as an oral disorder in the 1950s, the significance of this condition in causing widespread debility in the oral cavity and its propensity for malignant transformation (at 7.3% the highest in the group of potentially malignant oral disorders) has been well recognized.  The nature of the excess collagen in the tissues has been the subject of investigation since its discovery. Studies using standard light microscopy supplemented with special stains for collagen reported findings suggesting altered collagen. ,,,,, Electron microscopic (EM)/scanning electron microscopic (SEM) studies did not shed much light on the nature of collagen apart from identification of submicroscopic damage to collagen ends. ,,,,, Immunohistochemistry (IHC) and culture studies proliferation in the last two decades were largely responsible for conclusively identifying the type, content, and position of collagen in the disorder. ,,, In order to understand the pathogenesis of the disease, the identification of the collagen type and content is necessary. Over deposition of unaltered collagen would indicate an overstimulation of normal fibroblasts with probable reduced or absent degradatory collagenase control. On the other hand, altered collagen would point towards emergence of a phenotypically different fibroblast. The mode of deposition and collagen subtype would also indicate the reasons for the apparent irreversibility of the changes. For an understanding of the collagen deposition in the OSF, it is necessary to understand the distribution of collagen in the normal oral mucosa.
Collagen forms the structural network of the connective tissue component of the oral mucosal and is present in a fibrillar form. Collagen in the normal mucosa extends from the lamina propria which extends for varying depths in to the muscle layer. There are two different types of collagen organization in the normal oral mucosa. The upper connective stroma (lamina propria) contains a loose reticular network mainly composed of collagen types III and IV while collagen type I predominates in the deeper stroma.  Collagen I and III are major fibrillar components of the normal oral mucosa present in a ratio of 4:1 in the submucosa. Collagen fiber organization and fiber size vary topographically in the oral mucosa. For instance, in the buccal and gingival mucosa, collagen fiber bundles are thicker, less wavy, and of larger diameter in the deeper layers. They tend to be thinner, more wavy, and of smaller diameter towards the surface (near the epithelial junction). This difference is absent in the palate and rugae areas where the mucosa tends to be tightly adherent to underlying bone. In the palate and rugal regions, the fibril diameter generally remains constant.  Moreover, in contrast to extraoral skin, the collagenous network of intraoral submucosa does not contain elastic fibers and directly connects the lamina propria with the periosteum of jaws and hard palate.
The present review attempts to collate information from the literature with respect to the nature of collagen in the potentially malignant disorder of oral submucous fibrosis.
| Materials and Methods|| |
The PRISMA protocol for systematic analysis of literature was followed in the collection of data.  Medicine databases like PubMed, Pubget, ResearchGate, Ovid, and Medline were accessed using the search words "oral submucous fibrosis," "collagen subtypes in oral submucous fibrosis," "collagen I and III in oral submucous fibrosis," and "collagen in oral submucous fibrosis." The data from these studies were categorized based on the investigation modes into histopathological (standard light microscopic studies with or without special stains), EM/SEM, biochemical, IHC, culture studies, and genetic assays. The nature of collagen in the disorder with respect to content, type, and distribution was noted and analyzed. The rating of the publications being indexed (PubMed/Medline) noted and analyzed.
| Results|| |
A total of 63 acceptable articles were obtained from the search. Following the protocol and removal of duplicates a final of 30 articles were deemed satisfactory to be included in the review [Figure 1]. Though numerous studies have been conducted and reported on OSF, the search was limited to those involving the nature of collagen in the disorder. Of the 30 publications, 5 were in non-PubMed indexed publications but found acceptable due to the quality and content of the study. The predilection of the disorder to occur in the Asian continent (linked to the socially acceptable habit of areca nut consumption) prompted us to analyze the geographical distribution of the studies. Eight studies originated from across Europe, eight from the Chinese mainland, and the rest from the Indian subcontinent. The studies were further subdivided based on the methodology used for assessment of the nature of collagen in the disorder. Six studies were conducted using standard histological techniques including H& E and various connective tissue stains, six analyzed collagen birefringence by polarizing microscopy, and four studies estimated collagen biochemically. Advanced methodologies employed included EM/SEM studies (6), IHC (3), cultures of fibroblasts (4), and genetic evaluation (2). [Table 1] presents the relevant data.
|Figure 1: PRISMA protocol followed for analysis and collection of literature|
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|Table 1: Details of studies in literature on collagen in oral submucous fibrosis|
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| Discussion|| |
The methodologies employed in the assessment of the nature of collagen indicate the complexities of the disorder. Since collagen is aubiquitious component of the connective tissue its assessment in isolation is rather difficult. The mode of formation and deposition of collagen results in an intricate interlaced framework of different subtypes of collagen fibers with intervening extracellular matrix (ECM) components. Thus, tissue localization and mapping tend to be diffused and generalized. Yet the composite picture in most studies analyzed, indicate a definitive shift, and rearrangement of collagen subtypes and deposition patterns. Comparison of the fibrosis in OSF with that occurring in the normal mucosa and mucosal reaction due to non-specific irritation and inflammation reveals parallels in the initial stages of the reactions but establishment of a different collagen type and deposition in the progressive stages of the disease.
Histology (Light Microscopy)
The earliest reported histological studies on OSF were by Pindborg JJ and coworkers.  The connective tissue observations included increased dense collection of collagen fibers, juxta-epithelial hyalinization in advanced cases, variable inflammatory infiltration, edema, and decreased vascularity with advancing grades. These have largely been corroborated in subsequent reports with the exception of juxta-epithelial hyalinization. This feature seems to be intensely variable and only a few cases of advanced OSF have been reported with this finding. The hyalinization is absent in the early stages of the disease. Use of connective tissue stains has revealed variable results. In their initial studies on OSF, Pindborg and Sirsat (1967)  described densely thickened collagen and uneven staining pattern when stained with Mallory stains and Weigert's Resorcin Fuchsin, especially in the deeper parts of the mucosa, but with no abrupt tinctorial change. Hamner et al. demonstrated abnormal juxta-epithelial connective tissue in OSF using standard stains, indicating a probable alteration in the collagen. As the disease progressed, the connective tissue lost its fibrillar staining pattern and became amorphous. In another study on OSF using van Gieson stain for collagen, increased staining in the submucosa was found compared with the lamina propria and overall increased staining than in normal oral mucosa. The authors interpreted this observation as an indication that the initiation of fibrosis seems to be occurring from the deeper layers upwards, contrary to established perception of fibrosis being initiated from the juxta-epithelial region.  Muscle changes and involvement in OSF have been evaluated histologically using Masson Trichrome stains. There were variable depths of penetration of the muscle tissue by fibrosis ranging from no involvement to almost total replacement of muscle by the fibrotic process.  This probably indicates the aggressiveness of the deposition process. In yet another study involving muscle in OSF degenerative changes in the form of loss of striations, edema and atrophy were reported. 
The application of digitization in image analyzes is now an established procedure in histopathology. Morphometric analyzes of OSF revealed lack of quantifiable collagen in Grade I with a substantial increase in Grade II and III stages.  Our own studies, in contrast, revealed a gradual increase in stages with enough quantifiable material in Grade I, especially when compared with normal oral mucosa.
The histological observations in humans seem to be reflected in experimental animals. In a study, OSF like changes were induced in rats by painting of the buccal mucosa with areca nut extracts. Histologically the changes mimicked those seen in humans and were irreversible.  Our own studies in the experimental induction of OSF in Sprague-Dawley rats with areca nut extracts corroborate these observations.
Histological studies in OSF have served to identify and highlight the pathology but are by no means objective. The variations in stain technology and the observations thereon seem to highlight the issue. Nevertheless they are the primary investigative modes in the assessment of the disorder and important in establishing the progression of the disease.
The use of polarization in assessment of fibrosis has been enthusiastic in OSF especially in the Indian subcontinent. [22-28] Three parameters seem to have been evaluated in the assessment, quantification of collagen, assessment of collagen subtypes, and distribution of collagen in the connective area. In a polarizing microscope, collagen fibers are of two broad types, thin and thick. The former are mostly collagen type III with loosely arranged fibrils and numerous interfibrillar spaces while the latter are mostly collagen type I with thicker more mature fibers and less interfibrillar spaces. Though the distribution in normal oral mucosa is variable, thin fibers tend to be concentrated in the lamina propria and upper half of the submucosa while the thick fibers tend to be present in the deeper layers. Colorization varies with types of filters used. Thin fibers reflected as yellowish, green or greenish gray, and thick fibers as yellowish orange or orangish red. A common observation in all the studies seems to be substantially increased thick fiber content with advancing grades of the lesion, replacement of the thin fibers by thick fibers in the deeper layers and a more orangish red group of fibers in advanced grades. Correlated with collagen subtypes, it indicates predominance of collagen I at the expense of collagen III subtypes.
Polarizing microscopy even with application of image analyzes softwares is at best a quasi-objective attempt and by no means specific and reproducible. The variability of the colors and their interpretation is vast and the observations fraught with gross errors. Nevertheless, the information gleaned adds to the general pool of the nature of collagen in the disorder and indicates replacement of the more immature fibers with mature thick cross-linked fibers.
Electron microscopic (EM) and scanning electron microscopic (SEM) studies reveal structural changes at the submicroscopic level and are important in understanding the cellular changes existing in the disorder. An initial assessment of collagen fibers in OSF and betel nut chewer's mucosa revealed collection of amorphous deposits of crystalloid material intercellularly and between the fibrils. Dense bundles of collagen fibers were found next to these amorphous masses. These findings have not been replicated in later studies. Though the nature of the amorphous and crystalloid deposits was said to be unknown it is probable that the material represented ECM components that contribute to the density of the collagen deposits.  Further investigations did not reveal any significant variation in the type of collagen, most agreeing that the collagen type I being quantitatively more than other subtypes. ,,, No morphological alterations in collagen structure was reported. Three-dimensional assessment of connective tissue cores of collagen fibers using SEM showed progressively patchy degenerative cores with advancing grades of the disorder. This is probably linked to lack of remodeling capacity in the lesions secondary to decreased vascularity, depleted collagenase, and excess fibers. 
Lack of consistency in changes as observed by the EM/SEM may be due to interpretation errors. The thick masses of collagen probably do not lend themselves to EM scrutiny similar to observations under the light microscope.
An analysis of the ECM remodeling in OSF immunohistochemically revealed that in the early stage of OSF collagen type III along with certain ECM molecules (tenascin, perlecan, and elastin) were enhanced in the lamina propria and submucosal layer. In the intermediate stage, elastin was extensively and irregularly deposited around muscle fibers, together with the above-mentioned molecules. In the advanced stage, all those ECM molecules decreased and were entirely replaced by collagen type I only. Their gene expression levels were varied with the progression of fibrosis. The authors conclude that it is clear ECM remodeling steps in OSF is similar to each phase of usual granulation tissue formation and maturation. 
Kaur et al. analyzed colligin/HSP47 and col I protein in OSF. Colligin/HSP47 is a 47KDa stress protein which acts as a chaperone for collagen and plays a vital role in folding and assembling collagen. Overexpression of colligin and collagen type I proteins was observed in 70% and 65% of OSF cases, respectively. The conclusion, based on the data, was that the increased levels of colligin in OSF may contribute to the deposition of collagen and consequent increased fibrosis in the oral submucosa in OSF lesions.
In another IHC study of the distribution of procollagen type III and collagen type VI in OSF, the authors reported a loss of stainable procollagen type III and collagen type VI in the fibrotic zones of oral submucous fibrosis compared to normal oral mucosa. It was also observed that procollagen type III and collagen type VI in OSF were expressed in a specific pattern which allows a clear differentiation between fibrotic areas and adjacent apparently normal connective tissue stroma. They presumed that loss of procollagen type III indicated a probable predominance of collagen type I in collagen fiber bundles and attributed the clinical trismus in advanced cases to this change. Interestingly, the IHC findings provided evidence that the process of fibrosis starts in the deeper subepithelial connective tissue stroma and not close to the subepithelial basement membrane.
We analyzed collagen I and III immunohistochemically in OSF patients and attempted to morphometrically map the areas occupied by both the collagen types. The ratio of collagen I:III in initial cases of OSF was almost similar to normal oral mucosa but was found to significantly increased in progressive and advanced stages indicating a definitive tilt towards collagen type I. The phenomenon of fibrosis originating from the deeper layers and progressing to the epithelial junction was also corroborated by our findings. 
There seems to be a consensus based on IHC studies that increased fibrosis is primarily due to replacement of collagen III by collagen I and that the process is initiated from the deeper layers of the connective tissue.
Biochemical estimation of collagen
Attempts to biochemically analyze collagen in OSF have yielded predictable results. In a series of studies Anuradha et al., analyzed collagen content of OSF tissues in humans and in those from experimentally induced OSF in rats and found total tissue collagen increased compared with to the normal. This was corroborated by Huang et al. who reported increased collagen content in tissue samples of OSF patients as compared with skin and normal oral mucosa. The authors also reported that the glycine content of collagen in OSF was found to be lesser than the other groups. A replication of this biochemical alteration has not been further reported.
The results of the biochemical analyzes add to the pooled data corroborating the evidence that collagen in OSF is quantitatively more and qualitatively normal.
The nature of collagen deposited in tissue is a direct function of the fibroblast phenotype. Factors initiating the phenotypic change and deposition of collagen are best studied by culture studies. In OSF, these have concentrated on two aspects of the disorder; the nature of the fibroblast from the lesion and the reaction of normal fibroblasts to stimulation by areca nut, the causative factor associated with the disorder. ,,, In the earliest studies, areca nut extracts were found to have definitively stimulated collagen synthesis in fibroblast cultures. A 10 mg/dl concentration of areca nut extract was found to increase collagen synthesis by 150% in fibroblast cultures.  In yet another study, a 1.5-fold increase in collagen production in OSF fibroblasts was detected in cultures. The ratio of alpha 1(1): Alpha 1(2) collagen chains was 3:1 in OSF compared with 2:1 for type I collagen aclear indication of the quantitative and qualitative changes in collagen deposition. 
In another study, F3 fibroblastic cell lines (capable of producing more collagen I and III) as compared to F1 (normal fibroblasts) were found in OSF in contrast to normal oral mucosa. The induction of the phenotypic change in the fibroblasts leaning towards induction of cell lines capable of producing more stable and collagenase resistant collagen seems to be increasingly recognized. This induction is most likely initiated by components of the areca nut. 
The need to understand the cellular and genetic changes in the fibroblasts in OSF has become more relevant with increasing attempts to control the progression of the disorder. Most have centered on the fibroblast-ECM interaction during the process of collagen deposition. Studies on the nature type and induction of collagen formation are rather limited. , The association of OSF and polymorphisms of six collagen-related genes, collagen 1A1 and 1A2 (COL1A1 and COL1A2), collagenase-1 (COLase), transforming growth factor 1 (TGF-1), lysyl oxidase (LYOXase), and cystatin C (CST3) was examined between patients with low and high exposure to betel quids. (Chiu et al.). They found that the genotypes associated with the highest OSF risk for collagen 1A1, collagen 1A2, collagenase-1, transforming growth factor1, lysyl oxidase, and cystatin C were CC, AA, TT, CC, AA, and AA, respectively, for the low-exposure group, and TT, BB, AA, CC, GG, and AA, respectively, for the high-exposure group. A trend was noted for an increased risk of OSF with increasing number of high-risk alleles for those with both high and low exposures for betel quid. The cell selection mechanism of oral fibroblasts is proposed to explain the effect of the modification of cumulative betel quid exposure on the risk profiles of collagen-related genes.  These results imply that susceptibility to OSF could involve multigenic mechanisms modified by the betel quid-exposure dose.
the mode of collagen deposition involves a complex interplay of fibroblasts-cytokines-ECM components in OSF, the nature of the collagen deposited is increasingly becoming clear based on the studies investigated. Irritation to the oral mucosa caused by the components of the areca nut quid initiate a predictable inflammatory response from the tissues. The course and resolution of this response determines the final outcome of the disorder. Induction of fibroblast lines that produce more type I collagen and are resistant to degradation during the process of remodeling seems to be key to the change in the resultant fibrosis. In summation the following features are notable in the nature of collagen in OSF:
- Based on the histological, biochemical, and immunochemical studies, collagen in OSF seems to be essentially normal but increased quantitatively.
- Initial grades of OSF show similar pattern of deposition of collagen subtypes I and III as seen in normal oral mucosa, but the advancing grades show replacement in part or whole of collagen III by type I.
- Collagen deposition progresses from the deeper layers of the connective tissue and subsequently involves the juxta-epithelial regions.
- Experimentally induced OSF in rats using areca nut extracts essentially mirror changes seen in humans indicating a strong -causal relationship.
- Muscle degeneration is common due to replacement of muscle fibrosis with collagen.
- The change in the collagen deposition is initiated by induction of a phenotypic change in fibroblasts. Fibroblast lines more resistant to collagenase degradation and producing collagen type I are predominant.
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