Quantification of inflammatory, angiogenic, and fibrous components of reactive oral lesions with an insight into the pathogenesis

V Vasanthi; Bose Divya; Ramya Ramadoss; P Deena; Ramesh K Annasamy; Krishnan Rajkumar

doi:10.4103/jomfp.jomfp_138_21



ORIGINAL ARTICLE

Year : 2022 \| Volume : 26 \| Issue : 4 \| Page : 600

Quantification of inflammatory, angiogenic, and fibrous components of reactive oral lesions with an insight into the pathogenesis

V Vasanthi¹, Bose Divya¹, Ramya Ramadoss², P Deena¹, Ramesh K Annasamy¹, Krishnan Rajkumar¹
¹ Department of Oral Pathology and Microbiology, SRM Dental College, SRMIST, Chennai, Tamil Nadu, India
² Department of Oral Biology, Saveetha Dental College, SIMATS, Chennai, Tamil Nadu, India

Date of Submission	08-May-2021
Date of Decision	23-Mar-2022
Date of Acceptance	03-Jun-2022
Date of Web Publication	22-Dec-2022

Correspondence Address:
Ramya Ramadoss
Department of Oral Biology, Saveetha Dental College, SIMATS, Chennai, Tamil Nadu
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jomfp.jomfp_138_21

Abstract

Background: Reactive oral lesions pose diagnostic difficulties as they mimic each other clinically. A definitive diagnosis is made based on the histopathological presentation of this group of lesion. Stromal microenvironment is the key to the sequence of the stages of these lesions. Stringent quantification of each component of the stroma is important to understand the pathogenesis. The aim is to evaluate inflammation, angiogenesis, and fibrosis in the reactive group of lesions through quantitative analysis.
Materials and Methods: Blocks of irritation fibroma, inflammatory fibrous hyperplasia, pyogenic granuloma, and normal mucosa were retrieved from the archives and Hematoxylin and Eosin (H&E) and Masson Trichrome staining were done. The severity of inflammation, epithelial thickness, collagen proportionate area, integrated density of collagen, Mean Vascular Area (MVA), Mean Vascular Perimeter (MVP), and Mean blood vessel percentage area (MBVPA) were analysed quantitatively using Image J software version 1.8. The pattern of rete ridges at the epithelium-connective tissue interface was analysed qualitatively.
Results: Inflammatory fibrous hyperplasia presented with severe inflammation (60%). Mean Vascular Percentage Area (MVPA) and Mean Vascular Perimeter (MVP) were increased in pyogenic granuloma. The mean collagen proportionate area and the integrated density of collagen were found to be more in irritation fibroma (64.47%, 2519638.01 ± 810471.58 μm²). The epithelial thickness was highest in inflammatory fibrous hyperplasia (62.71 ± 18.86 μm).
Conclusion: Reactive oral lesions are histologically distinct, yet they exhibit considerable overlap depending on the stage of the lesion. A morphometric quantitative exploration of the individual pathogenic components may aid in specific diagnosis.

Keywords: Angiogenesis, fibrosis, inflammation, quantification, reactive oral lesions

How to cite this article:
Vasanthi V, Divya B, Ramadoss R, Deena P, Annasamy RK, Rajkumar K. Quantification of inflammatory, angiogenic, and fibrous components of reactive oral lesions with an insight into the pathogenesis. J Oral Maxillofac Pathol 2022;26:600

How to cite this URL:
Vasanthi V, Divya B, Ramadoss R, Deena P, Annasamy RK, Rajkumar K. Quantification of inflammatory, angiogenic, and fibrous components of reactive oral lesions with an insight into the pathogenesis. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Mar 24];26:600. Available from: https://www.jomfp.in/text.asp?2022/26/4/600/364785

Introduction

Oral tissues are constantly exposed to unabated external and internal stimuli, causing developmental, inflammatory, cystic, metabolic, infectious, and neoplastic pathologies. Reactive oral lesions are the most common group of lesions that develop in response to chronic irritation or trauma and invigorate exuberant tissue response. Irritation fibroma, pyogenic granuloma, peripheral giant cell granuloma, epulis fissuratum, inflammatory papillary hyperplasia, and inflammatory fibrous hyperplasia are frequently reported reactive oral lesions. They are non-neoplastic growths but sometimes may exhibit clinical resemblance to neoplastic lesions making the diagnosis challenging.^[1]

Chronic inflammatory immune response to chronic irritation is characterised by simultaneous remodelling and repair of tissues. The key to remodelling commences with the migration of inflammatory immune regulatory cells, proliferation of vascular endothelial cells, fibroblasts, and extracellular matrix synthesis. Cytokines, growth factors, and angiogenic factors play a definitive role in the repair process.^[2] Mast cells and macrophages have been reported to contribute to the pathogenesis of reactive oral lesions through their effects on endothelial cells and fibroblasts. They synthesise increased amounts of basic Fibroblast Growth Factor (bFGF) to be released into the extracellular matrix and also promote neovascularisation through pro-angiogenic cytokines primarily Vascular Endothelial Growth Factor (VEGF). Mast cell mediator, namely tryptase, promotes fibroblast activation, collagen deposition, and fibrosis.^{[3],[4],[5],[6],[7]} During the initial stage of the lesion, inflammatory cell infiltration, vascular proliferation, and appearance of myofibroblasts and type III collagen can be observed in response to the injury and repair. When the lesions mature, the connective tissue consists of densely packed type I collagen with no inflammation and the myofibroblasts disappear, suggestive of healing.^[8]

Clinically, reactive oral lesions appear as painless, pedunculated, or sessile overgrowths with colours ranging from the normal colour of the mucosa to red or gray ulcerated surface, depending on the duration of lesion and the type of injury.^[9] They have diverse histological features due to the variation in connective tissue response to the intensity of stimuli, ranging from highly vascularised, loose connective tissue in pyogenic granuloma to densely packed, well-organised connective tissue in fibroma.^[10]

To the best of our knowledge, there have been no studies quantitatively evaluating the inflammatory, angiogenic, and fibrotic components of these lesions. The knowledge about the composition of connective tissue is essential to arrive at a correct histopathological diagnosis. Therefore, the aim of the current study was to quantitatively analyse the inflammatory, angiogenic, and fibrotic components of irritation fibroma, inflammatory fibrous hyperplasia, pyogenic granuloma, and normal mucosa using image analysis software.

Materials and Methods

This retrospective study utilised formalin-fixed paraffin-embedded (FFPE) blocks of 30 irritation fibroma, 30 inflammatory fibrous hyperplasia, 30 pyogenic granuloma, and ten normal mucosa retrieved from the archives of the Department of Oral Pathology and Microbiology. Hematoxylin & Eosin (H&E) and Masson Trichrome staining were done and photomicrographs were captured in three high-power fields using a digital camera. All the captured images were standardised and calibrated in Image J software version 1.8 using a micrometer image with the unit of the measurement set as μm. The following morphologic parameters were analysed using the tools and plug-ins of the software.

Quantification of inflammatory component

The inflammatory cell counting was counted manually with the help of a cell counter > plug in > Image J. The severity of inflammation was evaluated among different groups based on the following criteria:^[11]

Mild: less than 100 inflammatory cells per field

Moderate: 100 to 250 inflammatory cells per field

Severe: more than 250 inflammatory cells per field.

Quantification of angiogenic component

The blood vessels were traced with the free hand tool to measure their area and perimeter and their mean values were calculated as Mean Vascular Area (MVA) and Mean Vascular Perimeter (MVP) respectively. Mean blood vessel percentage area (MBVPA) was calculated from the total area and blood vessel area in percentage.^[12] [Figure 1]

Figure 1: Measurement of Blood Vessel Area using Image J

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Quantification of fibrotic component

Original images were converted into Reg/Green/Blue (RGB) images and then deconvoluted using the colour deconvolution plugin. The threshold tool was then used to highlight the collagen fibres from the background tissue. Collagen Proportionate area refers to the proportion of collagen stained area to the total area in the region of interest for each image, which is expressed in %.^[13] The integrated density of collagen refers to the product of area and mean intensity of collagen stain.^[14] [Figure 2]

Figure 2: Thresholded collagen stained area (appears red) using colour deconvolution plug-in >> Image J

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Epithelial thickness

Epithelial thickness was measured from the most superficial point of the epithelium to the junction between epithelium and connective tissue at three different points by selecting Analyse > Measure tool and then the mean was calculated. The nature of rete ridges at the epithelium connective tissue interface was analysed qualitatively.^[15],[16] [Figure 3]

Figure 3: Pattern of rete ridges at the epithelium-connective tissue interface

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Statistical analysis

Data analysis was done using the Statistical Package for the Social Sciences (SPSS®) software version 21. Mean was calculated for all the parameters and non-parametric tests were used as the data was not normally distributed. Statistical analysis was performed using Analysis of Variance (ANOVA) and Kruskal Wallis to analyse continuous variables. A P value ≤ 0.05 was considered statistically significant.

Results

When the severity of inflammation was compared among different groups [Table 1], it was observed that the majority of the irritation fibroma cases (75%) had mild inflammation whereas the majority of pyogenic granulomas (70%), followed by inflammatory fibrous hyperplasia presented with severe inflammation (60%). Of the reactive group of lesions, Mean Vascular Area (MVA) was highest in pyogenic granuloma (12434.49 μm²), followed by inflammatory fibrous hyperplasia (5993.58 μm²) and irritation fibroma (3191.98 μm²), respectively [Table 2]. The difference was found to be statistically significant among the groups (P < 0.05). Mean Vascular Percentage Area (MVPA) and Mean Vascular Perimeter (MVP) were significantly increased in pyogenic granuloma (23.40% and 134.3 μm) than inflammatory fibrous hyperplasia (13.49% and 66.2 μm) and irritation fibroma (6.98% and 17.4 μm).

Table 1: Comparison of inflammatory components among different groups

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Table 2: Comparison of angiogenic components [Mean Vascular Area (MVA), Mean Vascular Perimeter (MVP), and Mean Vascular Percentage Area (MVPA)] among different groups

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[Table 3] represents the comparison of parameters like collagen proportionate area, integrated collagen density, and epithelial thickness among different groups. The mean collagen proportionate area and the integrated density of collagen were found to be more in irritation fibroma (64.47%, 2519638.01 ± 810471.58 μm²) when compared to other lesions. The epithelial thickness was highest in inflammatory fibrous hyperplasia (62.71 ± 18.86 μm), followed by pyogenic granuloma (53.90 ± 25.37 μm), irritation fibroma (30.73 ± 19.70 μm).

Table 3: Comparison of fibrotic component and epithelial thickness among different groups

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The most prominent rete ridge pattern was a box-shaped pattern in irritation fibroma, sawtooth pattern in inflammatory fibrous hyperplasia, whereas in pyogenic granuloma fused rete ridges were frequently observed. Various other patterns of rete ridges observed in inflammatory fibrous hyperplasia were camel foot (3.3%), church-spire (3.3%), and antler-like pattern (3.3%) and in pyogenic granuloma, an arborising pattern was seen (25%).

Discussion

Inflammatory, angiogenic, and fibrotic components were studied in the present study as the stromal microenvironment is vital for the development of reactive oral lesions. Reactive lesions of the oral cavity share common etiology such as chronic irritation from local factors, ill-fitting dentures, food impaction, and common clinical presentation. Even though they mimic clinically, they exhibit histological differences at different stages of development. The predominantly vascular lesion like pyogenic granuloma may mature and develop into a fibrous lesion in the later stages.^[17],[18]

Irritation fibroma is the reactive hyperplasia of the connective tissue occurring as a result of the chronic repair process. The collagen fibres may exhibit radiating pattern or circular pattern based on the degree of trauma and site of the lesion.^[19] Inflammatory fibrous hyperplasia presents depending on the extent of vascularity, collagenisation, and the presence or absence of inflammation. The fibroblast component may vary from paucicellular, bland to fine spindle-shaped cells in most lesions.^[20]

Preliminary reaction in response to long-standing stimuli is characterised by infiltration of defense cells in the connective tissue. Inflammatory cells ingress in the order of neutrophils, macrophages, plasma cells, and lymphocytes. Aggregation of these cells leads to a release of chemical mediators which can cause damaging effects on the extracellular matrix, resulting in vasodilatation, increased vascular permeability, hyperplasia, and neovascularisation, eventually replaced by fibroblastic proliferation.^{[21],[22],[23]} The activation and degranulation of mast cells is indispensable for the sequelae of events involved in the pathogenesis of reactive oral lesions. Tumor Necrosis Factor from mast cells causes migration of leukocytes and chronic inflammation. Chymase and tryptase from mast cells facilitate fibroblast proliferation and type I collagen synthesis.^[24] According to our findings, the severity of inflammation was high in pyogenic granulomas, followed by inflammatory fibrous hyperplasia and irritation fibroma. Hunasgi et al.^[25] reported increased intensity of inflammatory infiltrates in pyogenic granuloma than fibroma. This was attributed to the presence of increased estrogen and progesterone levels in periodontal tissues. Estrogen leads to increased proliferation of gingival fibroblasts and gingival inflammation.

Pyogenic granuloma, recently called lobular capillary hemangioma, mimics a tumour. VEGF and bFGF are the activators of angiogenesis that promote the lesion. VEGF is considered to be a potent mitogenic factor for endothelial cells and induces microvascular permeability. TSP-1 and angiostatin are the inhibitors of angiogenesis that result in regression of the lesion. Other vascular factors such as connective tissue growth factor, angiopoietin-1, angiopoietin-2, Tie-2, ephrinB2, ephrinB4, and decorin also have been reported by investigators to be involved in profound inflammation and angiogenesis in the pyogenic granuloma.^[26],[27] In the current study, MVPA and MVP were increased in pyogenic granuloma than inflammatory fibrous hyperplasia and irritation fibroma. This is consistent with the findings of Hunasgi et al.^[25] and Joy Thomas Vara et al.^[28] Increased vascularity in pyogenic granuloma may be due to a modified vascular response to local irritational stimuli. Joy Thomas Vara et al. studied the vascular and inflammatory index and reported high expression of VEGF in the pyogenic granuloma. Tissue hyperplasia has been demonstrated to occur due to increased levels of oestrogen and progesterone, triggering a neovascular response.^[29]

Collagen proportionate area is a quantitative indicator of the amount of collagen deposited in proportion to the total biopsy area. It has been established as an accurate measure of fibrosis and an independent predictor of clinical outcomes in non-alcoholic fatty liver disease. Collagen proportionate area was found to be more in irritation fibroma (64%) than in other lesions. The fibrotic events may be attributed to the functional fibroblasts activation by fibrogenic cytokines such as tumour necrosis factor, platelet-derived growth factor, and basic fibroblast growth factor.^[24],[30] Epithelial thickness was increased in inflammatory fibrous hyperplasia compared to other lesions. This may be due to the cytokines released by the inflammatory cells, resulting in the acanthosis and pseudoepitheliomatous hyperplasia of the overlying epithelium.^[21],[31]

The findings of the current study have to be validated in a larger sample with other special stains like Picrosirius red, which can highlight the natural birefringence of collagen when viewed under a polarised light microscope. There is a need to evaluate other reactive oral lesions to get an in-depth insight into the pathogenesis.

Conclusion

Reactive oral lesions exhibit histopathological differences despite the similarities in other aspects. The stromal microenvironment displays qualitative pathological changes which are imperative in the treatment of reactive lesions. A morphometric quantitative exploration of the individual pathogenic components may aid in specific follow-up care to prevent recurrence.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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