|Year : 2022 | Volume
| 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|
Department of Oral Biology, Saveetha Dental College, SIMATS, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| 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 Feb 3];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.
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. 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.,,,, 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.
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. 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.
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:
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. [Figure 1]
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 %. The integrated density of collagen refers to the product of area and mean intensity of collagen stain. [Figure 2]
|Figure 2: Thresholded collagen stained area (appears red) using colour deconvolution plug-in >> Image J|
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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., [Figure 3]
|Figure 3: Pattern of rete ridges at the epithelium-connective tissue interface|
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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 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.,
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. 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.
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.,, 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. According to our findings, the severity of inflammation was high in pyogenic granulomas, followed by inflammatory fibrous hyperplasia and irritation fibroma. Hunasgi et al. 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., 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. and Joy Thomas Vara et al. 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.
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., 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.,
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.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Naderi NJ, Eshghyar N, Esfehanian H. Reactive lesions of the oral cavity: A retrospective study on 2068 cases. Dent Res J (Isfahan) 2012;9:251-5.
Wynn TA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest 2007;117:524-9.
Aghbali AA, Akbarzadeh A, Kouhsoltani M. The role of macrophages and eosinophils in reactive lesions of the oral cavity. J Oral Maxillofac Pathol 2018;22:147.
] [Full text]
Shekar S, Angadi PV. Quantification of mast cells in reactive oral lesions – A clue to the morphologic diversity. Indian J Health Sci Biomed Res 2019;12:123-6. [Full text]
Murata M, Hara K, Saku T. Dynamic distribution of basic fibroblast growth factor during epulisformation: An immunohistochemical study in an enhanced healing process of the gingiva. J Oral Pathol Med 1997;26:224-32.
Kapoor P, Deshmukh RS. VEGF: A critical driver for angiogenesis and subsequent tumor growth: An IHC study. J Oral Maxillofac Pathol 2012;16:330-37.
] [Full text]
Sheethal HS, Kn H, Smitha T, Chauhan K. Role of mast cells in inflammatory and reactive pathologies of pulp, periapical area and periodontium. J Oral Maxillofac Pathol 2018;22:92-7.
] [Full text]
Nakamura F, Fifita SF, Kuyama K. A study of oral irritation fibroma with special reference to clinicopathological and immunohistochemical features of stromal spindle cells. Int J Oral-Med Sci 2005;4:83-91.
Kolte, Kolte RA, Shrirao TS. Focal fibrous overgrowths: A case series and review of literature. Contemp Clin Dent 2010;1:271-4.
Mighell AJ, Robinson PA, Hume WJ. Histochemical and immunohistochemical localization of elastic system fibres in focal reactive overgrowths of oral mucosa. J Oral Pathol Med 1997;26:153-8.
Jakubowska K, Koda M, Kisielewski W, Kańczuga-Koda L, Famulski W. Prognostic significance of inflammatory cell response in patients with colorectal cancer. Oncol Lett 2019;18:783-91.
Murgod VV, Kale AD, Angadi PV, Hallikerimath S. Morphometric analysis of the mucosal vasculature in oral submucous fibrosis and its comparison with oral squamous cell carcinoma. J Oral Sci 2014;56:173-8.
Ramadoss R, Krishnan R, Vasanthi V, Bose D, Vijayalakshmi R, Padmanabhan R, et al
. New insights for consummate diagnosis and management of oral submucous fibrosis using reactive and reparative fibrotic parameter derived algorithm. J Pharm Bioall Sci 2021;13:S323-32.
Chen SH, Peng CY, Chiang IP, Lai HC, Lee CJ, Su WP, et al
. Comparison of collagen proportionate areas in liver fibrosis quantification between chronic hepatitis B and C. Medicine 2016;95:e4736.
Rathore AS, Gupta A, Shetty DC, Kumar K, Dhanapal R. Redefining epithelial characterization in oral submucous fibrosis using morphometric analysis. J Oral Maxillofac Pathol 2017;21:36-40.
] [Full text]
Madke B, Doshi B, Khopkar U, Dongre A. Appearances in dermatopathology: The diagnostic and the deceptive. Indian J Dermatol Venereol Leprol 2013;79:338-48.
] [Full text]
Peralles PG, Viana AP, Azevedo AL, Pires FR. Gingival and alveolar hyperplastic reactive lesions: Clinicopathological study of 90 cases. Braz J Oral Sci 2006;5:1085-9.
Awange DO, Wakoli KA, Onyango JF, Chindia ML, Dimba EO, Guthua SW. Reactive localised inflammatory hyperplasia of the oral mucosa. East Afr Med J 2009;86:79-82.
Barker DS, Lucas RB. Localised fibrous overgrowths of the oral mucosa. Br J Oral Surg 1967;5:86-92.
Brierley DJ, Crane H, Hunter KD. Lumps and bumps of the Gingiva: A pathological miscellany. Head Neck Pathol 2019;13:103-13.
Hasturk H, Kantarci A. Activation and resolution of periodontal inflammation and its systemic impact. Periodontol 2015;69:255-73.
Sangle VA, Pooja VK, Holani A, Shah N, Chaudhary M, Khanapure S. Reactive hyperplastic lesions of the oral cavity: A retrospective survey study and literature review. Indian J Dent Res 2018;29:61-6.
] [Full text]
Kadeh H, Saravani S, Tajik M. Reactive hyperplastic lesions of the oral cavity. Iran J Otorhinolaryngol 2015;27:137-44.
Kadeh H, Derakhshanfar G, Saravani S. Comparative study of mast cell count in oral reactive lesions and its association with inflammation. Turk Patoloji Derg 2016;32:22-6.
Hunasgi S, Koneru A, Vanishree M, Manvikar V. Assessment of reactive gingival lesions of oral cavity: A histopathological study. J Oral Maxillofac Pathol 2017;21:180.
] [Full text]
Babu B, Hallikeri K. Reactive lesions of oral cavity: A retrospective study of 659 cases. J Indian Soc Periodontol 2017;21:258-63.
] [Full text]
Yuan K, Jin YT, Lin MT. Expression of Tie-2, angiopoietin-1, angiopoietin-2, ephrinB2 and EphB4 in pyogenic granuloma of human gingiva implicates their roles in inflammatory angiogenesis. J Periodontal Res 2000;35:165-71.
Vara JT, Gurudu VS, Ananthaneni A, Bagalad BS, Kuberappa PH, Ponnapalli HP. Correlation of vascular and inflammatory index in oral pyogenic granuloma and periapical granuloma-An insight into pathogenesis. J Clin Diagn Res 2017;11:ZC25-8.
Marla V, Shrestha A, Goel K, Shrestha S. The histopathological spectrum of pyogenic granuloma: A case series. Case Rep Dent 2016;2016:1323798.
Buzzetti E, Hall A, Ekstedt M, Manuguerra R, Misas MG, Covelli C, et al
. Collagen proportionate area is an independent predictor of long-term outcome in patients with non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2019;49:1214-22.
Sarangarajan R, Vaishnavi Vedam VK, Sivadas G, Krishnaraj R, Sarangarajan A, Shanmugam KT. Pseudoepitheliomatous hyperplasia: Relevance in oral pathology. J Int Oral Health 2015;7:132-6.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]