Ultrastructural Changes of Collagen in Different Histopathological Grades of Oral Submucous Fibrosis.

BACKGROUND AND OBJECTIVES: Oral submucous fibrosis (OSMF) is a potentially malignant disorder and a crippling condition of oral mucosa. It is usually seen in adults with areca nut chewing habit, which is characterized by changes in the connective tissue fibers leading to stiffness of the mucosa and restricted mouth opening. Patients with severe cases have distinct difficulties in chewing, swallowing, and speaking. It predominantly occurs in Indians and other population of the Indian subcontinent with a prevalence of 0.2%-0.5%. The potentiality of malignant transformation associated with OSMF is much higher with a range of 4.5%-7.6%. This article is about the ultrastructural changes pertaining to collagen and with respect to the histopathological grades of OSMF.
MATERIALS AND METHODS: A total of 20 biopsy samples from clinically diagnosed OSMF subjects and 5 healthy controls were collected. After obtaining the biopsy, the specimens were divided into two halves, of which one was studied under light microscope and the other half was observed under transmission electron microscope.
RESULTS: Of the 20 subjects in the study group, most of the subjects were in the 20-29 years age group with an overall male predilection (19 subjects). Four subjects were in grade 1, nine were in grade 2, and seven were in grade 3. Collagen showed sparse to dense fibrosis and normal to thick collagen bundles, and some subjects showed encroachment of collagen into the blood vessels.
CONCLUSION: This study showed definitive changes with respect to collagen in the OSMF samples compared to the controls. The changes were found to be increasing with the progression of the disease to the higher grades. The changes were pertaining to the collagen and were drawing toward an increased fibrosis of the connective tissue, which compresses the blood vessels. Hence, the state of hypoperfusion and subsequent epithelial atrophy can be considered in the progression of disease to the higher grades.

INTRODUCTION

The history of oral submucous fibrosis (OSMF) begins in 1952 with the first case that was reported by Schwartz and he termed it as “Atrophica idiopathica mucosae oris.” One year after, Joshi reported the first case in the Indian subcontinent and he termed it as “Submucous fibrosis of the palate and pillars.” Many other names emerged later on, i.e., diffuse oral submucous fibrosis (Lal 1953), idiopathic scleroderma of the mouth (Su 1954), idiopathic palatal fibrosis (Rao 1962), and sclerosing stomatitis (Behl 1962).[12]

The prevalence of OSMF seemed to increase in the Indian subcontinent with an overall rate of 0.2%–0.5% with a definite male predilection.[345678] The etiology of OSMF is strongly related to betel nut chewing along with local irritants such as tobacco, spicy foods, and alcohol, and systemic conditions such as autoimmunity, vitamin, and nutritional deficiencies also can contribute to OSMF.[139101112131415] Paymaster described the development of slow growing squamous cell carcinoma in one-third of patients with OSMF. Currently, the rate of malignant transformation is estimated as 4.5%–7.6%.[1416]

The onset of OSMF begins at the fauces followed by buccal and labial areas, resulting in reduction of mouth opening. It is usually associated with initial symptoms such as burning sensation of the oral mucosa, inability to eat spicy food, appearance of blisters, ulcerations or generalized inflammation of the oral mucosa, excessive salivation, defective gustatory sensation, and dryness of mouth. Later the oral mucosa becomes blanched and slightly opaque with appearance of vertical fibrous bands, which causes inability to open mouth, fixation, and shortening or even deviation of the uvula and soft palate.[123]

Histopathologically the changes begin at the level of lamina propria with the accumulation of hyalinized collagen beneath the basement membrane. On the basis of the amount and nature of the subepithelial collagen in the specimens from the OSMF, patients can be grouped into four grades: very early, early, moderately advanced, and advanced. Other changes include presence of normal, dilated, and constricted vessels seen in combination in the same section along with the presence of inflammatory cells mainly lymphocytes and plasma cells.[117]

Many authors have conducted ultrastructural studies using transmission electron microscope to evaluate the changes in collagen in OSMF. But none of the studies were reported correlating the ultrastructural changes with respect to different grades of OSMF. Hence, in this article we are evaluating the ultrastructural changes with respect to histopathological grades of OSMF.

MATERIALS AND METHODS

The study group composed of 20 patients with clinical signs and symptoms of OSMF and 5 subjects in the control group who were devoid of any symptoms and any other systemic disorders. An incisional biopsy was performed after obtaining the written consent from all subjects. The subjects were divided into three groups on basis of histopathologic grading based on the criteria that were modified from the original criteria given by Pindborg and Sirsat[18] as given later:

Grade 1—Mild hyalinization of juxtaepithelial area, plump young fibroblasts, dilated or congested blood vessels along with the presence of inflammatory cells mostly mononuclear lymphocytes, eosinophils, and few or occasional plasma cells.

Grade 2—Moderately hyalinized collagen, less marked fibroblastic response, presence of mostly fibrocytes, constricted blood vessels, and inflammatory exudate consisting of mostly lymphocytes and plasma cells with occasional eosinophils.

Grade 3—Complete collagen hyalinization with depletion of fibroblasts in those areas, blood vessels that are completely obliterated or narrowed with the presence of inflammatory cells such as lymphocytes and plasma cells.

RESULTS

Total study group was composed of 20 subjects of OSMF with different histopathological grades and 5 samples in the control group. The age of the samples in the study group was ranging from 22 to 64 years with the mean age of 35.05 years and the control groups were composed of ages ranging from 22 to 28 years with the mean age of 25.8 years [Table 1].

Table 1

Distribution of the subjects with respect to age group and histopathological grade of oral submucous fibrosis

Age group (in years)	Grade 1	Grade 2	Grade 3	Total
20–29	1 (5%)	4 (20%)	3 (15%)	8 (40%)
30–39	2 (10%)	1 (5%)	3 (15%)	6 (30%)
40–49	0	3 (15%)	0 (0%)	3 (15%)
50–59	0	1 (5%)	1 (5%)	2 (10%)
60–69	1 (5%)	0	0	1 (5%)
Total	4 (20%)	9 (45%)	7 (35%)	20 (100%)

The experimental group was divided into 3 groups based on the defined histopathological grading criteria: grade 1 composed of 4 cases, grade 2 composed of 9 cases, and grade 3 composed of 7 cases [Table 1].

Electron microscopic analysis

The ultrastructural analysis of collagen in the upper half of the tissue section revealed sparsely arranged fibers in 12 (60%) of the subjects comprising 4 (100%) subjects of grade 1, 5 (55.55%) subjects of grade 2, 3 (42.85%) subjects of grade 3, and rest of the samples were showing densely arranged fibers [Figure 1]. Variation in the fiber bundle diameter was seen in 19 (95%) subjects, comprising 3 (75%) subjects of grade 1, 9 (100%) subjects of grade 2, and 7 (100%) subjects of grade 3 [Table 2].

Figure 1

Thick numerous collagen bundles. Some of the fibers are ruptured and showing spaces in between (×6510)

Table 2

Ultrastructural changes in collagen with respect to the histopathological grades of oral submucous fibrosis in the upper half of the connective tissue

Ultrastructural features	Grade 1 (n = 4)	Grade 2 (n = 9)	Grade 3 (n = 7)
Sparsely populated fibers	4 (100%)	5 (55.55%)	3 (42.85%)
Densely populated fibers	0	4 (44.44%)	4 (57.14%)
Variation in fiber bundle diameter	3 (75%)	9 (100%)	7 (100%)

Collagen fibers in the lower half of the specimen revealed normal pattern in only 2 (50%) subjects of grade 1 in comparison to the thick bundles that were seen in 18 (90%) subjects, consisted of 2 (50%) subjects of grade 1, 9 (100%) subjects of grade 2, and 7 (100%) subjects of grade 3. Nineteen (95%) subjects showed variation in the fiber bundle diameter, comprising 3 (75%) subjects of grade 1, 9 (100%) subjects of grade 2, and 7 (100%) subjects of grade 3 [Table 3] [Figure 2]. The encroachment of collagen fibrils into the blood vessels was seen in 3 subjects of grade 3 [Table 4] [Figure 3].

Table 3

Ultrastructural changes in collagen with respect to the histopathological grades of oral submucous fibrosis in the lower half of the connective tissue

Ultrastructural features	Grade 1 (n = 4)	Grade 2 (n = 9)	Grade 3 (n = 7)
Normal bundles	2 (50%)	0	0
Thick bundles	2 (50%)	9 (100%)	7 (100%)
Variation in fiber bundle diameter	3 (75%)	9 (100%)	7 (100%)

Figure 2

Cross section of collagen with variation in the bundle diameter (×6510)

Table 4

Showing encroachment of collagen fibers with respect to the histopathological grades of oral submucous fibrosis

Ultrastructural feature	Grade 1 (n = 4)	Grade 2 (n = 9)	Grade 3 (n = 7)
Encroachment of fibers to blood vessels	0	0	3 (42.85%)

Figure 3

Deeper connective tissue showing compression of the blood vessels with encroachment of the collagen fibers (*) in between the endothelial cells (#) and few inflammatory cells are also seen (×5580)

DISCUSSION

The pathway to the cause of OSMF is clearly attributed to the abnormally increased production and reduced degradation of collagen in the connective tissue.[910] It could be due to the increase in ratio of F3 to F1 fibroblasts in response to the arecaidine, a product formed by the hydrolysis of arecoline component of the areca nut along with copper, which results in fibroblast stimulation.[2919] Once the collagen is formed, it gets further stabilized by the increased cross-linking by the tannin component of the areca nut along with reduced collagenase activity.[9]

The significant findings in this study were the changes in thickness, pattern, variation in fiber bundle diameter, and the encroachment of collagen fibers into the adjacent blood vessels. The lamina propria was divided into upper and lower half and the morphology of the collagen was studied. In the upper half, the density of collagen fibers and the variation in the bundle diameter were studied. All the controls and grade 1 subjects have showed sparsely arranged collagen fibers, but only 5 (55.55%) subjects of the grade 2 and 3 (42.85%) subjects of grade 3 have shown the same feature. Similar findings were reported by van Wyk et al.[20]; they have documented that there is a thin zone that is adjacent to the basement membrane and is sparsely populated with individual collagen fibrils and loosely arranged groups of fibrils running parallel to the epithelial connective tissue junction. Binnie et al.[21] have reported that the normal pattern of uniformly sized collagen fibrils that are gathered together in bundles was replaced by fine immature fibrils in an interfibrillar matrix. This occurrence of immature fibrils could be the reason for the occurrence of sparsely populated fibrils at the superficial areas of the lamina propria.[2021]

In contrast to the sparse arrangement of the collagen fibers in the superficial half of lamina propria, 4 (44.44%) subjects of grade 2 and 4 (57.41%) of the grade 3 subjects have shown dense accumulation of collagen. It could be due to the complete hyalinization and fibrosis of the connective tissue with the progression of the disease to the advanced stages.[1]

Compared to the upper half, the thickness of the collagen bundles was found to be more in the lower half of the lamina propria. In 2 (10%) subjects, normal bundles were seen, and all were in grade 1 constituting 50% of all subjects in the group. All subjects from grade 2 and 3 were showing thick bundles, constituting of about 18 (90%) subjects from the study population. The predominance of the thick bundles in the lower half of the lamina propria could be due to the initiation of the fibrosis at the deeper lamina propria, which was proved by various histochemical studies.[22]

In the control specimens, the diameter of the collagen fiber bundles was seemed to be uniform. But 95% of the diseased subjects in the upper half showed variation in fibril diameter within the same bundle, which constituted all the subjects of grade 2 and grade 3, and 3 (75%) subjects of grade 1. In the lower half, 19 (95%) subjects (i.e., 3 [75%] subjects of grade 1, 9 [100%] subjects of grade 2, and 7 [100%] subjects of grade 3) have shown similar features, which were in agreement with van Wyk et al.[20] and Binnie et al.[21] Because OSMF is primarily a collagen metabolic disorder caused by increased production and reduced degradation of collagen, the variation in the fiber bundle diameter could be due to increase in the presence of mature collagen and synthesis of new immature collagen at an abnormal pace.[392021]

Three (15%) subjects of grade 3 (42.85% in the group) have shown encroachment of collagen fibrils into the blood vessel. Most of the other subjects have shown increased fibrosis and compression of the blood vessels by the collagen bundles. Similar findings were reported by van Wyk et al.[20] and could be the reason for the reduction in the epithelial perfusion causing the ischemic atrophy, which ultimately leads to the epithelial atrophy.[1820]

CONCLUSION

This study was the first of its kind in which the samples of OSMF were divided into three groups based on the histopathological criteria and studied under the transmission electron microscope to evaluate the ultrastructural changed associated with respect to the structural and architectural changes of collagen. With the aforementioned features associated, it is clearly evident that the structural and architectural changes in collagen are more predominant in the advanced stages of OSMF than that we could observe under the light microscope; henceforth, the clinicians should aim at the early intervention and frequent follow-up to prevent the advancement of this condition to a carcinomatous change.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.