Expression pattern of VEGF, MMP 9,CD34 and CD68 in Orofacial Granulomatosis, Inflammatory Fibroepithelial Hyperplasia, and Oral Focal Mucinosis

Expression pattern of VEGF, MMP 9,CD34 and CD68 in Orofacial Granulomatosis, Inflammatory Fibroepithelial Hyperplasia, and Oral Focal Mucinosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression pattern of VEGF, MMP 9,CD34 and CD68 in Orofacial Granulomatosis, Inflammatory Fibroepithelial Hyperplasia, and Oral Focal Mucinosis Pratibha Ramani, Reena Das, Jayakumar ND, Casilda Sushanthi, Abilasha Ramasubramanian, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8891946/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background Orofacial granulomatosis (OFG), inflammatory fibroepithelial hyperplasia (IFH), and oral focal mucinosis (OFM) share characteristics of chronic inflammation and tissue remodeling in response to local irritation or immune dysregulation within the oral cavity. Methods This study investigates the expression of VEGF, MMP-9, CD68, and CD34 in OFG, IFH, and OFM, aiming to identify potential overlapping etiological factors, inflammatory pathways, and clinical patterns. By examining these molecular markers, we seek to uncover possible commonalities in pathogenesis that may guide diagnosis, inform treatment approaches, and improve management strategies for these conditions. Results Case analyses included histopathological and immunohistochemical evaluations. In OFG, CD34, MMP-9, and CD68 expression were under 10%, with no VEGF detected. In contrast, IFH showed over 40% positivity for CD34 and VEGF among inflammatory cells, low CD68 (< 10%), and non-specific MMP-9 staining. OFM displayed less than 10% VEGF expression, minimal CD34 positivity (5%), and absent MMP-9 and CD68. Conclusions These findings suggest distinct biomarker expression patterns that reflect vascularity and inflammatory profiles, which could aid in diagnosis and management. Specifically, VEGF expression was moderate in IFH, minimal in OFG, and absent in OFM, indicating varying angiogenic activity. The prominent presence of CD34 and CD68 in OFG and IFH highlights immune cell involvement, while minimal MMP-9 suggests limited extracellular matrix remodeling. Overall, these results offer insights into the inflammatory and vascular characteristics of these oral conditions. Case Series Vascular Endothelial Growth Factor (VEGF) Matrix metalloproteinase-9 (MMP-9) Orofacial granulomatosis (OFG) CD38 Fibroepithelial hyperplasia mucinosis vascularization biomarkers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Gingiva is the most dynamic structure in oral cavity as it is subject to constant irritation from calculus, food impaction, ill-fitting restorations, trauma, and several iatrogenic factors and hence is the most common site for localized reactive hyperplastic lesions. It also has a rich complex vascularity which makes it an ideal candidate for manifestation for various systemic, infectious and idiopathic disease conditions [ 1 , 2 ]. Orofacial Granulomatosis (OFG) is a chronic inflammatory condition characterized by persistent swelling of the lips, face, and oral mucosa especially gingiva. It is associated with granulomatous inflammation, where clusters of immune cells (granulomas) form in the affected tissues. OFG is often considered a manifestation of underlying systemic conditions such as Crohn's disease CD sarcoidosis, but it can also present with no known underlying cause [ 3 ]. Inflammatory Fibroepithelial Hyperplasia (IFH), commonly known as epulis when occurring in the gingiva, is a benign proliferation of fibrous connective tissue and overlying epithelium that arises as a reactive response to chronic irritation or inflammation. This condition may present as drug-induced gingival hyperplasia, denture-related epulis, or other forms of localized tissue overgrowth. These lesions are typically firm, painless, and histologically characterized by hyperplastic connective tissue [ 4 ]. Oral Focal Mucinosis (OFM) is a rare, benign condition predominantly affecting the gingiva and is characterized by the localized accumulation of mucin within the connective tissue. It typically manifests as a painless, slow-growing nodule [ 5 ]. Although the exact cause is unclear, OFM is considered a localized reactive process, akin to IFH. While OFG primarily affects the lips, its occasional presentation in the gingiva, coupled with the predominant gingival manifestation of IFH and OFM, creates challenges in their differential diagnosis. These conditions share overlapping clinical features such as swelling or nodular growths in the oral cavity and involve inflammatory responses, suggesting possible shared underlying mechanisms or triggers. Both inflammatory fibroepithelial hyperplasia and OFM are considered reactive lesions, meaning they occur as a response to local stimuli or irritation [ 6 ]. This reactive process might also play a role in the pathogenesis of OFG. In the context of orofacial granulomatosis, inflammatory fibroepithelial hyperplasia, and oral focal mucinosis, the investigation of inflammatory and vascular markers can provide insights into the pathogenesis and help in diagnosis and management. Vascular Endothelial Growth Factor (VEGF) is a key regulator of angiogenesis (formation of new blood vessels) and vascular permeability. VEGF levels are often elevated in chronic inflammatory conditions and can contribute to the vascular changes seen in these oral pathologies, particularly in conditions involving significant tissue remodelling and swelling [ 7 , 8 ]. MMP 2 and MMP 9 are Enzymes involved in the degradation of extracellular matrix components. Their activity is often upregulated in chronic inflammation and may contribute to tissue remodelling seen in inflammatory fibroepithelial hyperplasia and granulomatous condition [ 9 ]. CD34 serves as an important marker for endothelial cells in reactive gingival lesions, reflecting the extent of angiogenesis and vascularity. It also helps in identifying and assessing the role of fibroblasts and provides valuable diagnostic information regarding the vascular and cellular characteristics of the lesions [ 10 ]. CD68, a macrophage marker, highlights the involvement of macrophage-driven inflammatory pathways, particularly in granulomatous and reactive conditions. This study aims to assess the expression patterns of VEGF, MMP-9, CD68, and CD34 across cases of OFG, IFH, and OFM to determine if these markers reveal overlapping etiological factors, shared inflammatory and vascular pathways, or specific clinical characteristics that link these conditions. By identifying potential commonalities in the pathogenesis of these disorders, this research may improve our understanding of the molecular mechanisms driving these conditions, guiding diagnosis and helping refine management strategies for patients with similar oral inflammatory lesions. Ultimately, this investigation may contribute to establishing a more integrated approach to the diagnosis and treatment of chronic inflammatory and proliferative conditions in the oral cavity. Materials and Methods Case Selection and Clinical Procedure Three clinical cases were selected for this study based on their presentation and preliminary diagnoses: Orofacial Granulomatosis (OFG), Inflammatory Fibroepithelial Hyperplasia (IFH), and Oral Focal Mucinosis (OFM). Each case was evaluated clinically and histopathologically. Case 1: A 38-year-old female patient presented with swelling, pain, and bleeding of the gingiva. Clinical examination revealed multiple nodular lesions. Following oral hygiene measures (scaling and polishing), the lesions were surgically excised and submitted for histopathology (Fig. 1 ). Upon histopathological evaluation, the case was diagnosed as IFH. Case 2: A 37-year-old female with a history of hypothyroidism reported bleeding and multiple nodular swellings of both upper and lower gingiva. The patient underwent routine scaling, followed by excision of affected tissues. Specimens were submitted for histopathological examination to confirm the diagnosis (Fig. 2 ). The case was diagnosed as OFG. Case 3: A 29-year-old female presented with a localized rubbery swelling on the palatal aspect of the gingiva. The lesion was surgically excised, and the specimen was analyzed histologically (Fig. 3 ). After histopathological evaluation, the case was diagnosed as OFM. Histopathological analysis Excised tissues were processed using standard histopathological techniques and stained with hematoxylin and eosin. Histopathological examination confirmed diagnoses and provided insight into cellular architecture, inflammatory infiltrates, and tissue characteristics specific to each condition. Histopathological examination of the excised tissues confirmed diagnoses and provided insight into cellular architecture, inflammatory infiltrates, and tissue characteristics specific to each condition. Histological sections of IFH, showed parakeratinized stratified squamous epithelium with pseudoepitheliomatous hyperplasia, a dense fibrous connective tissue stroma with numerous capillaries, chronic inflammatory infiltrate, and areas of hemorrhage. Connective tissue stroma in OFG exhibited non-caseating granulomas composed of epithelioid cells and multinucleated giant cells admixed with a chronic inflammatory infiltrate. In OFM, mucinous areas with stellate and spindle-shaped fibroblasts within loose mucinous connective tissue, minimal inflammatory cells, and moderate vascularity were observed. Alcian blue staining confirmed mucin presence. Immunohistochemical staining Immunohistochemistry (IHC) was performed on tissue sections (3–4 µm thick) using the Dako EnVision FLEX + Mouse, High pH kit (Dako, Denmark) following the manufacturer’s protocol. The primary antibodies targeted CD34, CD68, MMP9, and VEGF to assess vascularization, inflammation, and tissue remodeling within the samples. Tissue sections were mounted on Poly-L-Lysine-coated slides to promote adherence. Slides were incubated overnight at 50°C, then deparaffinized in three changes of xylene (10 minutes each) and rehydrated through graded ethanol series. Antigen retrieval was conducted by immersing slides in an EDTA buffer solution (pH 9.0) and heating them under pressure in an electric cooker for 20 minutes, followed by gradual cooling to ambient temperature. To block endogenous peroxidase activity, slides were treated with Dako EnVision FLEX Peroxidase-Blocking Reagent for 30 minutes. After blocking, sections were rinsed twice in Tris-buffered saline (TBS, pH 7.4) for 3 minutes each to prepare for antibody incubation. Slides were then incubated with primary antibodies for CD34, CD68, MMP9, and VEGF for 1 hour at room temperature, followed by two additional TBS washes to remove excess antibody. Following primary antibody incubation, slides were treated with a secondary antibody for 30 minutes and washed twice in TBS. Chromogenic detection was achieved by applying 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate to the sections for 5 minutes, resulting in a brown precipitate indicating positive antigen expression. Slides were counterstained with Harris hematoxylin, air-dried, and permanently mounted using DPX (Dibutylphthalate Polystyrene Xylene) for visualization under a light microscope. The scoring for IHC was done using the following labelling index, % of IHC + Labelled cells 0 = 0% 1 = 60% [ 11 ] Slides were independently reviewed by two pathologists to assess the expression levels of CD34, CD68, MMP9, and VEGF. Results As presented in Table 1 , no VEGF expression was detected, and less than 10% positivity was observed for CD34, and CD68 in the case of Orofacial Granulomatosis (Fig. 4 ). For Inflammatory Fibroepithelial Hyperplasia (Fig. 5 ), more than 40% positivity was noted for CD34 and VEGF among inflammatory cells, while less than 10% of cells were positive for CD68, and MMP9 exhibited non-specific staining. In Oral Focal Mucinosis (Fig. 6 ), less than 10% of cells showed positivity for VEGF, approximately 5% for CD34, and no positivity was observed for MMP9 or CD68. Table 1 Immunohistochemical report of the 03 cases Case No. VEGF MMP9 CD34 CD68 165 − 22 Orofacial granulomatosis Negative 30% positivity > 10% positivity 181 − 21 Inflammatory fibroepithelial hyperplasia 40% positivity > 10% positivity 225-21Oral Focal mucinosis < 15% focal positivity in inflammatory cell Negative < 5% positivity Negative Discussion The expression pattern of VEGF,CD 34, MMP 9, and CD 68 seen in our cases shows the complex process and highlights the potential implications. VEGF is a critical signalling protein involved in the formation of new blood vessels and the regulation of vascular permeability [ 12 ]. In the context of orofacial granulomatosis (OFG), inflammatory fibroepithelial hyperplasia, and oral focal mucinosis (OFM), VEGF have been considered in the pathogenesis and progression of these conditions [ 13 ]. OFG is characterized by chronic inflammation, which often leads to tissue swelling and the formation of granulomas. VEGF may contribute to vascularization of these granulomas, to support the immune response within the granuloma VEGF increases vascular permeability, leading to leakage of fluid from blood vessels into the surrounding tissues [ 12 ]. The non expression pattern of VEGF in our case may indicate tha t this case could indicate a long standing granuloma. A negative or low expression of VEGF might indicate that the inflammatory response in OFG is not associated with significant new blood vessel formation. This could potentially result in less pronounced tissue swelling or less severe edema compared to cases where VEGF is highly expressed [ 14 ]. Granulomas in OFG are often sustained by an active inflammatory response and the formation of new blood vessels. If VEGF is low or absent, the granulomas might be less vascularized, possibly leading to a different composition or structure of the granulomas. This could influence the chronicity of the granulomas, potentially making them more stable or less prone to changes over time [ 15 ]. VEGF is also involved in increasing vascular permeability, which contributes to the inflammatory exudate and edema seen in inflamed tissues. A lack of VEGF might result in a different inflammatory profile, with potentially fewer exudative features and a different pattern of immune cell infiltration. This could lead to a less aggressive inflammatory response in OFG, possibly affecting the severity of the disease. The clinical features of OFG, such as lip swelling, facial edema, and oral mucosal changes, could be less pronounced if VEGF expression is low. The degree of tissue swelling and the extent of visible granulomatous changes might be reduced in cases with negative VEGF expression. Patients with low VEGF might present with milder symptoms or a different distribution of lesions compared to those with high VEGF expression [ 16 ].The absence of VEGF expression could be used as a diagnostic marker to differentiate between different subtypes or severities of OFG. It might also help distinguish OFG from other granulomatous conditions where VEGF is typically upregulated. In cases where VEGF is not driving the disease process, anti-angiogenic therapies targeting VEGF might not be effective. Instead, other therapeutic strategies targeting different aspects of the inflammatory response or granuloma formation might be more appropriate [ 17 ]. Inflammatory fibroepithelial hyperplasia involves the proliferation of fibrous connective tissue and overlying epithelium in response to chronic irritation or inflammation [ 18 ]. VEGF promotes angiogenesis in these hyperplastic tissues, supporting their growth and maintenance. The increased expression of VEGF in these lesions can lead to the development of a dense network of new blood vessels, which is often observed in hyperplastic tissues. These vascular changes can contribute to the persistence and expansion of the lesion [ 19 ]. In inflammatory fibroepithelial hyperplasia, < 40% positivity for Vascular Endothelial Growth Factor (VEGF) among inflammatory cells indicates that less than 40% of the inflammatory cells in the tissue express VEGF. <40% positivity suggests that VEGF expression is present but not extensive. This implies that angiogenesis, or the formation of new blood vessels, is occurring at a moderate level in the affected tissue. The limited VEGF expression indicates that while there is some vascular proliferation, it is not the predominant feature. The tissue may have a moderate degree of new blood vessel formation compared to conditions with high VEGF expression and that VEGF is contributing to the inflammatory process but not overwhelmingly. The moderate VEGF expression might reflect a balanced role in supporting tissue repair and inflammation, rather than driving excessive angiogenesis. While < 40% positivity indicates moderate VEGF expression, it helps to understand the extent of angiogenic activity in the lesion. This level of VEGF expression can provide insights into the balance between inflammation and tissue repair processes. Treatment strategies might need to address both the inflammatory and hyperplastic components of the lesion. OFM is characterized by the localized accumulation of mucin within the connective tissue. VEGF may play a role in the vascularization of these mucinous nodules, ensuring an adequate blood supply to the affected area. Although OFM is a rare condition, its reactive nature to local stimuli might involve the upregulation of VEGF, which facilitates angiogenesis and supports the mucinous changes within the oral mucosa. VEGF plays a crucial role in the pathogenesis of inflammatory fibroepithelial hyperplasia, and have limited expression in long standing granulomas and in OFM. Understanding VEGF's involvement in these conditions can provide insights into potential diagnostic markers and therapeutic targets. MMP 9 is an enzyme that plays a significant role in the remodeling of the extracellular matrix (ECM) by degrading type IV collagen, a major component of the basement membrane. MMP-9 is involved in various physiological and pathological processes, including inflammation, tissue repair, and tumor progression. In OFG, chronic inflammation leads to the formation of granulomas, which are aggregates of immune cells attempting to contain the inflammation. MMP-9 is upregulated in response to inflammatory cytokines and is involved in the remodeling of the ECM surrounding granulomas [ 20 ]. This remodeling is essential for granuloma maintenance and expansion, as MMP-9 facilitates the migration of immune cells to the site of inflammation by breaking down the ECM. MMP-9 contributes to the degradation of ECM components, leading to tissue remodeling. This process is often associated with the swelling and fibrosis seen in OFG. The increased activity of MMP-9 can result in the breakdown of the basement membrane, allowing immune cells to infiltrate the tissue and exacerbate inflammation. The non expression of MMP9 further reiterates that our case is a long standing lesion. In orofacial granulomatosis (OFG), < 10% focal positivity for Matrix Metalloproteinase-9 (MMP-9) means that MMP-9 expression is detected in less than 10% of the examined tissue areas. This level of expression provides specific insights into the role of MMP-9 in the disease. Here's an analysis < 10% focal positivity indicates that MMP-9 is expressed in only a small portion of the tissue. This suggests that its role in the pathology of OFG is limited or localized rather than widespread. The small areas showing MMP-9 positivity may reflect specific regions where extracellular matrix remodeling is occurring, but overall, MMP-9 may not be a major factor in the disease process. Granulomas in OFG are characterized by a collection of macrophages and other immune cells. Limited MMP-9 expression suggests that the remodeling of extracellular matrix components is not a prominent feature of the granulomas. Low MMP-9 positivity implies that matrix degradation or remodeling might not be a significant part of the inflammatory response in OFG, or that it occurs only in specific, localized areas. <10% positivity means MMP-9 is not a primary marker for OFG. Its limited expression suggests that other factors or pathways might be more relevant to the disease pathology. The low level of MMP-9 expression helps differentiate OFG from conditions with higher MMP-9 positivity, where matrix remodeling and tissue damage might be more prominent [ 21 ]. Since MMP-9 is not highly expressed, targeting MMP-9 may not be particularly effective for treating OFG. The focus of treatment might need to address other aspects of the inflammatory response or granuloma formation. Monitoring MMP-9 levels could be useful for assessing localized matrix remodeling but may not provide comprehensive insights into disease progression or response to treatment. Other biomarkers or clinical parameters should be considered for a complete evaluation. The low focal positivity of MMP-9 suggests that its role in OFG might be less significant compared to other conditions with more pronounced MMP-9 activity [ 22 ]. Research could focus on understanding why MMP-9 is minimally expressed and identify other key factors driving the disease. In inflammatory fibroepithelial hyperplasia, there is a reactive proliferation of fibrous tissue and epithelium in response to chronic irritation. MMP-9 plays a key role in this process by degrading the ECM, which allows for the expansion of the hyperplastic tissue. This degradation facilitates the migration and proliferation of fibroblasts and epithelial cells, contributing to the growth of the hyperplastic lesion. MMP-9 is often upregulated in chronic inflammatory conditions and is produced by various cells, including macrophages, neutrophils, and fibroblasts. In inflammatory fibroepithelial hyperplasia, the persistent inflammation and tissue irritation likely lead to increased MMP-9 expression, which further drives tissue remodeling and lesion progression [ 23 ]. In the context of inflammatory fibroepithelial hyperplasia, nonspecific expression of Matrix Metalloproteinase-9 (MMP-9) means that the enzyme is present, but its expression does not correlate strongly with specific pathological features of the condition. MMP-9 is involved in the breakdown of extracellular matrix components, which plays a role in tissue remodeling, inflammation, and repair. Nonspecific expression suggests that MMP-9 is involved in general tissue remodeling and matrix turnover rather than a targeted pathological process. Also that it may contribute to the overall remodeling of fibrous and epithelial tissues but is not specifically driving the hyperplastic changes.MMP-9 is often upregulated in various inflammatory conditions due to its role in tissue damage and repair. Nonspecific expression means that while MMP-9 is present, it is not uniquely associated with the inflammation in this specific condition. The enzyme's expression may reflect a broad inflammatory response rather than a specific contributor to the inflammatory or hyperplastic process in fibroepithelial hyperplasia [ 23 ]. Oral focal mucinosis is characterized by the localized accumulation of mucin within the connective tissue. MMP-9 may be involved in the ECM remodeling that occurs in response to this mucinous change. By degrading ECM components, MMP-9 could facilitate the formation and expansion of mucinous nodules [ 24 ]. In oral focal mucinosis, a negative result for Matrix Metalloproteinase-9 (MMP-9) indicates that MMP-9 is not expressed or detected in the tissue. MMP-9 is an enzyme involved in the breakdown of extracellular matrix components, and its expression is often associated with tissue remodeling, inflammation, and certain pathological conditions [ 25 ]. Non expression of MMP-9 suggests that there is little to no breakdown of extracellular matrix (ECM) components by this enzyme in the tissue. This implies that the pathological changes in oral focal mucinosis are not associated with significant ECM remodeling mediated by MMP-9. Since MMP-9 is involved in ECM degradation, its absence might indicate that mucin accumulation in oral focal mucinosis is not associated with extensive tissue remodeling or degradation of ECM proteins. The absence of MMP-9 expression highlights that inflammation in oral focal mucinosis may not involve significant ECM degradation or be driven by MMP-9-mediated processes. CD34 is a cell surface glycoprotein that functions as a marker of hematopoietic stem cells, endothelial cells, and progenitor cells involved in blood vessel formation. It is widely used as a marker for identifying and quantifying blood vessels, particularly in the context of angiogenesis [ 26 ]. CD34- positive blood vessels are often observed in areas of active inflammation and granuloma formation in OFG. The presence of CD34-positive vessels can be indicative of the extent of vascular involvement and the degree of angiogenesis associated with the inflammatory process in OFG as observed in our case. In orofacial granulomatosis (OFG), > 30% positivity for CD34 indicates that more than 30% of the examined tissue areas exhibit significant expression of CD34. CD34 is a cell surface glycoprotein commonly used as a marker for endothelial cells and hematopoietic progenitor cells [ 27 ]. CD34 positivity is often associated with endothelial cells and neovascularization. In OFG, > 30% positivity indicates significant vascular proliferation, suggesting that the granulomatous lesions are highly vascularized. This increased vascularization can contribute to tissue swelling and redness, typical of OFG [ 28 ]. The high percentage of CD34- positive areas reflects a high density of blood vessels in the affected tissue, which may be involved in the inflammatory response and the overall pathology of OFG. Granulomas in OFG may exhibit increased angiogenesis as part of the inflammatory response. CD34 positivity highlights that new blood vessel formation is a prominent feature of the granulomas. The presence of numerous blood vessels can contribute to the observed symptoms of OFG, such as swelling, pain, and erythema. High CD34 positivity can be indicative of active disease with significant inflammatory and vascular components. It helps in distinguishing OFG from other conditions with less pronounced vascular changes. Tracking CD34 positivity can be useful for monitoring disease activity and assessing responses to treatments aimed at reducing inflammation and angiogenesis. The high vascularization observed with > 30% CD34 positivity suggests that therapies targeting angiogenesis might be beneficial. Treatments that reduce blood vessel formation could help alleviate symptoms and potentially improve clinical outcomes [ 28 ]. Inflammatory fibroepithelial hyperplasia involves the proliferation of fibrous tissue and epithelium in response to chronic irritation. This process is often accompanied by increased blood vessel formation to support the growing tissue. CD34 is a key marker for identifying these new blood vessels. The degree of CD34 expression can provide insights into the level of angiogenesis occurring within the hyperplastic tissues. As part of the tissue's response to chronic irritation, CD34- positive endothelial progenitor cells may contribute to the repair and regeneration processes by promoting new blood vessel formation. This vascular support is crucial for the maintenance and growth of the hyperplastic lesion [ 29 ]. Oral focal mucinosis is characterized by the localized accumulation of mucin within the connective tissue. The formation of these mucinous nodules is often accompanied by the development of new blood vessels to support the tissue changes. Limited CD34 expression in our case can be used to assess the extent of vascularization within these nodules. Given that OFM is a reactive condition, the presence of CD34- positive blood vessels may reflect the tissue's response to local stimuli. CD68 is a glycoprotein primarily expressed on the surface of monocytes and macrophages, making it an important marker for identifying and studying these immune cells. CD68 is widely used as a marker to identify these macrophages within granulomatous lesions. The presence of CD68- positive macrophages in granulomas indicates their role in containing the inflammatory stimulus and in orchestrating the immune response. In OFG, macrophages play a critical role in sustaining the chronic inflammatory response by secreting pro-inflammatory cytokines and chemokines. CD68 expression is a marker of macrophage activation and can be used to assess the extent of macrophage involvement in the disease process. High levels of CD68- positive cells are indicative of active inflammation and granulomatous tissue formation [ 30 ]. In orofacial granulomatosis (OFG), > 10% positivity for CD68 suggests that more than 10% of the examined tissue areas exhibit significant expression of CD68. CD68 is a marker commonly associated with macrophages, which are key players in inflammation and immune responses. CD68 positivity indicates the presence of macrophages in the tissue. >10% positivity suggests a notable infiltration of macrophages within the granulomas of OFG, highlighting the role of these cells in the inflammation. Macrophages are integral to granuloma formation. Their significant presence supports the diagnosis of OFG, as these cells are crucial in the formation and maintenance of granulomatous inflammation. High levels of CD68 positivity point to an active inflammatory response in OFG. Macrophages play a central role in chronic inflammation and tissue response to irritants or foreign substances. The presence of macrophages suggests ongoing immune activation and response, which is a hallmark of granulomatous conditions.>10% positivity for CD68 is indicative of significant macrophage activity and can help confirm the presence of OFG. It supports the diagnosis by demonstrating the extent of inflammatory involvement. This level of CD68 positivity helps differentiate OFG from other oral lesions where macrophage infiltration might be less pronounced [ 31 ]. The significant presence of macrophages suggests that treatment strategies might need to focus on modulating the inflammatory response. Therapies that target macrophage activity or the inflammatory pathways they are involved in could be beneficial. In inflammatory fibroepithelial hyperplasia, chronic irritation leads to the proliferation of fibrous tissue. Macrophages, marked by CD68, are often present in these lesions as part of the chronic inflammatory response. They may contribute to tissue remodeling by releasing enzymes and growth factors that influence fibroblast activity and collagen production, leading to the thickening of the fibroepithelial tissue. Macrophages are also involved in tissue repair processes, where they help clear debris, secrete factors that promote healing, and modulate inflammation. The presence of CD68- positive macrophages in hyperplastic tissue could indicate ongoing tissue remodeling and repair in response to chronic irritation or inflammation [ 32 ]. In inflammatory fibroepithelial hyperplasia, > 10% positive for CD68 indicates that more than 10% of the examined tissue areas exhibit significant expression of CD68. >10% positivity for CD68 suggests that a substantial proportion of the inflammatory cells in the tissue are macrophages. The presence of macrophages is indicative of ongoing inflammation. These cells are involved in the inflammatory response, tissue repair, and the regulation of immune responses. Although OFM is a relatively benign condition, macrophages may still play a role in the reactive processes. CD68- positive macrophages could be involved in clearing cellular debris, responding to local stimuli, and possibly modulating the accumulation of mucin within the tissue. Even though OFM is not primarily an inflammatory condition, the presence of CD68-positive macrophages might be observed in cases where there is a mild or localized inflammatory response associated with the mucinous nodules. These macrophages could be involved in maintaining tissue homeostasis [ 33 ]. In our case negative expression of CD68 suggests that macrophages, or cells with macrophage-like properties, are not present in the tissue or are present in such low numbers that they are below the detection threshold and so there is minimal or no macrophage-driven inflammation in oral focal mucinosis. The negative CD68 result supports the idea that the condition is primarily due to mucin deposition rather than an inflammatory response involving macrophages Conclusion By analyzing these markers, clinicians and researchers can gain a better understanding of the underlying mechanisms driving these oral pathologies, potentially leading to more effective management and treatment strategies. Future scope/ clinical significance This study highlights the differential expression of VEGF, MMP-9, CD34, and CD68 across orofacial granulomatosis (OFG), inflammatory fibroepithelial hyperplasia, and oral focal mucinosis (OFM). Future research could validate these markers' diagnostic and prognostic utility in larger cohorts. Identifying thresholds for biomarker positivity may help refine diagnostic criteria, enabling earlier and more accurate differentiation between these conditions. Further investigation into VEGF and MMP-9 pathways may reveal novel therapeutic targets, particularly in cases with prominent angiogenesis and extracellular matrix remodeling.For conditions with low VEGF and MMP-9 expression, studies could explore alternative mechanisms driving pathogenesis and identify more suitable therapeutic approaches. Abbreviations OFG- Orofacial Granulomatosis OFM- Oral Focal Mucinosis VEGF- Vascular Endothelial Growth Factor MMP- Metallic metalloproteinase ESR- Erythrocyte Sedimentation Rate QFT- . Quantiferon TB gold Test ACE- Angiotensin- Converting Enzyme IOPA- Intraoral Periapical Radiograph LA- Local Anaesthesia IHC- Immunohistochemistry TBS- Tris Buffer Saline DAB- 3,30-diaminobenzidine tetrahydrochloride DPX- Dibutylphthalate Polystyrene Xylene ECM- Extra Cellular Matrix MMP- Matrix Metalloprotease Declarations Ethics approval and consent to participation Ethical approval and informed consent to participate was obtained from all of the participants in the study by Saveetha Dental College and Hospitals Institutional Ethics Committee (SDC-IHEC), Registered under Government of India, (ECR/1452/Inst/TN/2024). The study adhered to the Declaration of Helsinki. Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interest The authors declare that they have no competing interests Funding Not applicable Author’s contribution PR, RD, JND, CS, and AR contributed to conceptualization; PR, RD, AR contributed to methodology; RD, JND, CS, AR contributed to validation; RD, PR, JND, CS, NU, and MRM contributed to formal analysis; RD, PR and AR contributed in formal analysis, investigation and resources; GS and AR were involved in data curation; RD, PR and AR were involved in writing- original draft preparation, review and editing; RD, PR, JND, CS and AR involved in visualization; RD and PR involved in supervision. All authors read and approved the final manuscript. Acknowledgements The authors acknowledge Dr. N.M. Veeraiyan, Chancellor, and Dr. Deepak Nallaswamy SIMATS University for providing the extended essential facilities for the research work. The authors acknowledge Dr. Nisha Jaishree, MDS, Oral Pathology, SIMATS for helping in conducting the Research. Author’s information 1 Department of Oral Pathology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamilnadu- 600077 2 Department of Periodontology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India- 600077 3 Department of Oral and Maxillofacial Surgery, EHS, Fujairah Specialized Dental Center and Hospital, Fujairah, UAE References Suresh R, Ramadoss R, Krishnasamy N, Selvam SP, Sundar S, K H. Interplay of thyroid dysfunction and burning mouth syndrome: a case report. Egypt J Intern Med. 2025;37:57. https://doi.org/10.1186/s43162-025-00442-9. Theofilou VI, Pettas E, Georgaki M, Daskalopoulos A, Nikitakis NG. Localized juvenile spongiotic gingival hyperplasia: Microscopic variations and proposed change to nomenclature. 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Increased Levels of Circulating Angiogenic Cells and Signaling Proteins in Older Adults With Cerebral Small Vessel Disease. Front Aging Neurosci. 2021;13:711784. https://doi.org/10.3389/fnagi.2021.711784. Galdiero MR, Maio F, Arcoleo F, Boni E, Bonzano L, Brussino L, et al. Orofacial granulomatosis: Clinical and therapeutic features in an Italian cohort and review of the literature. Allergy. 2021;76:2189–200. https://doi.org/10.1111/all.14799. Shimizu Y. Mechanism underlying vascular remodeling in relation to circulating CD34-positive cells among older Japanese men. Sci Rep. 2022;12:21823. https://doi.org/10.1038/s41598-022-26089-y. Hassanpour M, Salybekov AA, Kobayashi S, Asahara T. CD34 positive cells as endothelial progenitor cells in biology and medicine. Front Cell Dev Biol. 2023;11:1128134. https://doi.org/10.3389/fcell.2023.1128134. Labrador AJP, Valdez LHM, Marin NRG, Ibazetta KAR, Chacón JAL, Fernandez AJV, et al. Oral granulomatosis with polyangiitis a systematic review. Clinical & Exp Dental Res. 2023;9:100–11. https://doi.org/10.1002/cre2.706. Sekar K, Ramanathan A, Khalid R, Mun KS, Valliappan V, Ismail SM. An unusual case of multiple primary tumours involving the long bone and oral cavity. Oral Maxillofac Surg. 2025;29:62. https://doi.org/10.1007/s10006-025-01356-0. Jiang C, Wang W, Chen Y-L, Chen J-H, Zhang Z-W, Li J, et al. Macrophage polarization and macrophage-related factor expression in hypertrophy of the ligamentum flavum. Eur Spine J. 2024. https://doi.org/10.1007/s00586-024-08513-1. Lehman JS, Sokumbi O, Peters MS, Bridges AG, Comfere NI, Gibson LE, et al. Histopathologic features of noninfectious granulomatous disorders involving the skin. Human Pathology. 2020;103:127–45. https://doi.org/10.1016/j.humpath.2020.05.008. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Apr, 2026 Reviews received at journal 28 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 24 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 23 Apr, 2026 Editor invited by journal 06 Apr, 2026 Editor assigned by journal 23 Feb, 2026 Submission checks completed at journal 23 Feb, 2026 First submitted to journal 23 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8891946","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631676705,"identity":"72e25e5b-4eb9-4849-949f-e3968a1f59d6","order_by":0,"name":"Pratibha Ramani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYLCCxAYoI6ECQjMT0MDYgNByBiLSTFALI0wLYxsRWsylDz9/8HCHXT7/tMPPHjycVydvcLz5+OOCGgZ5frEDWLVY9qUZNiSeSbaccTvN3CBx22HDDWeOJTbPOMZgOHN2AlYtBmcYgFramA0YbieYSSRuO8A4c0aOYTMPG0OCwW1cWtg/ArXUG8jfTv8mkTinzn7m/Pcfm3n+4dPCA7LlsIHB7RygLQ3Mif0SPIzNvG24tVj28BTOSDxz3MDwdk6ZRMKxw8n9PGmGs3n7JHD6xZyHfcPHnzuqDeRup2+T/FFTZ9vGfvjBZ55vNvL80jgchlUUCCRwSeDWMgpGwSgYBaMADgBqMmJ+/EJlbgAAAABJRU5ErkJggg==","orcid":"","institution":"Saveetha Institute of Medical And Technical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Pratibha","middleName":"","lastName":"Ramani","suffix":""},{"id":631676708,"identity":"a5ead1a2-144e-440d-a183-239228aae19b","order_by":1,"name":"Reena Das","email":"","orcid":"","institution":"Saveetha Institute of Medical And Technical 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09:58:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8891946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8891946/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108735089,"identity":"dd7bf668-b4e8-48b4-bc00-7c661cfecd98","added_by":"auto","created_at":"2026-05-07 20:00:01","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1886401,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple reddish swelling involving upper and lower labial gingiva of the premolar region\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/2d3cb9af4017d1ae370f7184.jpg"},{"id":108807070,"identity":"b0c01938-d64c-403e-ad15-90818892d2ac","added_by":"auto","created_at":"2026-05-08 15:30:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140833,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple nodular swellings in both upper and lower gingiva\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/cd0d38c36f1bf64fec019f21.jpg"},{"id":108806617,"identity":"6b9fdf24-392f-4ddd-a91f-5b30b5d91687","added_by":"auto","created_at":"2026-05-08 15:29:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107563,"visible":true,"origin":"","legend":"\u003cp\u003eWell defined pinkish swelling with nodular surface in the palatal aspect of right pre molar and molar gingiva\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/0905c1bd9255dce4a25804a9.jpg"},{"id":108735091,"identity":"b6c66598-b528-422b-ab7c-0bdfbbdbdbef","added_by":"auto","created_at":"2026-05-07 20:00:01","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2702226,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of CD34 and CD68 in the case of Orofacial Granulomatosis\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/86b5ad28c4845a9091c3a42f.jpg"},{"id":108735095,"identity":"cda3e619-5147-4b3e-b1aa-8513a30b7408","added_by":"auto","created_at":"2026-05-07 20:00:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2279973,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of CD34 and VEGF in the case of Inflammatory Fibroepithelial Hyperplasia\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/38bfa73af5dbc8fbd17d2868.jpg"},{"id":108807186,"identity":"215b7182-c1f3-47fb-b2ce-89c0d62f05a3","added_by":"auto","created_at":"2026-05-08 15:30:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2400844,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemistry of VEGF in Oral Focal Mucinosis\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/f521774f3c3b6921f02a0925.jpg"},{"id":108809945,"identity":"2e36bd73-d469-42ea-abc1-fb0261c271fe","added_by":"auto","created_at":"2026-05-08 15:56:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9748272,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8891946/v1/09063d97-61db-4e26-9824-cc8da09d87ae.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression pattern of VEGF, MMP 9,CD34 and CD68 in Orofacial Granulomatosis, Inflammatory Fibroepithelial Hyperplasia, and Oral Focal Mucinosis","fulltext":[{"header":"Background","content":"\u003cp\u003e Gingiva is the most dynamic structure in oral cavity as it is subject to constant irritation from calculus, food impaction, ill-fitting restorations, trauma, and several iatrogenic factors and hence is the most common site for localized reactive hyperplastic lesions. It also has a rich complex vascularity which makes it an ideal candidate for manifestation for various systemic, infectious and idiopathic disease conditions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Orofacial Granulomatosis (OFG) is a chronic inflammatory condition characterized by persistent swelling of the lips, face, and oral mucosa especially gingiva. It is associated with granulomatous inflammation, where clusters of immune cells (granulomas) form in the affected tissues. OFG is often considered a manifestation of underlying systemic conditions such as Crohn's disease CD sarcoidosis, but it can also present with no known underlying cause [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Inflammatory Fibroepithelial Hyperplasia (IFH), commonly known as epulis when occurring in the gingiva, is a benign proliferation of fibrous connective tissue and overlying epithelium that arises as a reactive response to chronic irritation or inflammation. This condition may present as drug-induced gingival hyperplasia, denture-related epulis, or other forms of localized tissue overgrowth. These lesions are typically firm, painless, and histologically characterized by hyperplastic connective tissue [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOral Focal Mucinosis (OFM) is a rare, benign condition predominantly affecting the gingiva and is characterized by the localized accumulation of mucin within the connective tissue. It typically manifests as a painless, slow-growing nodule [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Although the exact cause is unclear, OFM is considered a localized reactive process, akin to IFH. While OFG primarily affects the lips, its occasional presentation in the gingiva, coupled with the predominant gingival manifestation of IFH and OFM, creates challenges in their differential diagnosis. These conditions share overlapping clinical features such as swelling or nodular growths in the oral cavity and involve inflammatory responses, suggesting possible shared underlying mechanisms or triggers. Both inflammatory fibroepithelial hyperplasia and OFM are considered reactive lesions, meaning they occur as a response to local stimuli or irritation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This reactive process might also play a role in the pathogenesis of OFG.\u003c/p\u003e \u003cp\u003eIn the context of orofacial granulomatosis, inflammatory fibroepithelial hyperplasia, and oral focal mucinosis, the investigation of inflammatory and vascular markers can provide insights into the pathogenesis and help in diagnosis and management. Vascular Endothelial Growth Factor (VEGF) is a key regulator of angiogenesis (formation of new blood vessels) and vascular permeability. VEGF levels are often elevated in chronic inflammatory conditions and can contribute to the vascular changes seen in these oral pathologies, particularly in conditions involving significant tissue remodelling and swelling [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. MMP 2 and MMP 9 are Enzymes involved in the degradation of extracellular matrix components. Their activity is often upregulated in chronic inflammation and may contribute to tissue remodelling seen in inflammatory fibroepithelial hyperplasia and granulomatous condition [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CD34 serves as an important marker for endothelial cells in reactive gingival lesions, reflecting the extent of angiogenesis and vascularity. It also helps in identifying and assessing the role of fibroblasts and provides valuable diagnostic information regarding the vascular and cellular characteristics of the lesions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CD68, a macrophage marker, highlights the involvement of macrophage-driven inflammatory pathways, particularly in granulomatous and reactive conditions.\u003c/p\u003e \u003cp\u003eThis study aims to assess the expression patterns of VEGF, MMP-9, CD68, and CD34 across cases of OFG, IFH, and OFM to determine if these markers reveal overlapping etiological factors, shared inflammatory and vascular pathways, or specific clinical characteristics that link these conditions. By identifying potential commonalities in the pathogenesis of these disorders, this research may improve our understanding of the molecular mechanisms driving these conditions, guiding diagnosis and helping refine management strategies for patients with similar oral inflammatory lesions. Ultimately, this investigation may contribute to establishing a more integrated approach to the diagnosis and treatment of chronic inflammatory and proliferative conditions in the oral cavity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCase Selection and Clinical Procedure\u003c/h2\u003e \u003cp\u003eThree clinical cases were selected for this study based on their presentation and preliminary diagnoses: Orofacial Granulomatosis (OFG), Inflammatory Fibroepithelial Hyperplasia (IFH), and Oral Focal Mucinosis (OFM). Each case was evaluated clinically and histopathologically. Case 1: A 38-year-old female patient presented with swelling, pain, and bleeding of the gingiva. Clinical examination revealed multiple nodular lesions. Following oral hygiene measures (scaling and polishing), the lesions were surgically excised and submitted for histopathology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Upon histopathological evaluation, the case was diagnosed as IFH. Case 2: A 37-year-old female with a history of hypothyroidism reported bleeding and multiple nodular swellings of both upper and lower gingiva. The patient underwent routine scaling, followed by excision of affected tissues. Specimens were submitted for histopathological examination to confirm the diagnosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The case was diagnosed as OFG. Case 3: A 29-year-old female presented with a localized rubbery swelling on the palatal aspect of the gingiva. The lesion was surgically excised, and the specimen was analyzed histologically (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). After histopathological evaluation, the case was diagnosed as OFM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological analysis\u003c/h3\u003e\n\u003cp\u003eExcised tissues were processed using standard histopathological techniques and stained with hematoxylin and eosin. Histopathological examination confirmed diagnoses and provided insight into cellular architecture, inflammatory infiltrates, and tissue characteristics specific to each condition. Histopathological examination of the excised tissues confirmed diagnoses and provided insight into cellular architecture, inflammatory infiltrates, and tissue characteristics specific to each condition. Histological sections of IFH, showed parakeratinized stratified squamous epithelium with pseudoepitheliomatous hyperplasia, a dense fibrous connective tissue stroma with numerous capillaries, chronic inflammatory infiltrate, and areas of hemorrhage. Connective tissue stroma in OFG exhibited non-caseating granulomas composed of epithelioid cells and multinucleated giant cells admixed with a chronic inflammatory infiltrate. In OFM, mucinous areas with stellate and spindle-shaped fibroblasts within loose mucinous connective tissue, minimal inflammatory cells, and moderate vascularity were observed. Alcian blue staining confirmed mucin presence.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemical staining\u003c/h3\u003e\n\u003cp\u003eImmunohistochemistry (IHC) was performed on tissue sections (3\u0026ndash;4 \u0026micro;m thick) using the Dako EnVision FLEX\u0026thinsp;+\u0026thinsp;Mouse, High pH kit (Dako, Denmark) following the manufacturer\u0026rsquo;s protocol. The primary antibodies targeted CD34, CD68, MMP9, and VEGF to assess vascularization, inflammation, and tissue remodeling within the samples. Tissue sections were mounted on Poly-L-Lysine-coated slides to promote adherence. Slides were incubated overnight at 50\u0026deg;C, then deparaffinized in three changes of xylene (10 minutes each) and rehydrated through graded ethanol series. Antigen retrieval was conducted by immersing slides in an EDTA buffer solution (pH 9.0) and heating them under pressure in an electric cooker for 20 minutes, followed by gradual cooling to ambient temperature. To block endogenous peroxidase activity, slides were treated with Dako EnVision FLEX Peroxidase-Blocking Reagent for 30 minutes. After blocking, sections were rinsed twice in Tris-buffered saline (TBS, pH 7.4) for 3 minutes each to prepare for antibody incubation. Slides were then incubated with primary antibodies for CD34, CD68, MMP9, and VEGF for 1 hour at room temperature, followed by two additional TBS washes to remove excess antibody. Following primary antibody incubation, slides were treated with a secondary antibody for 30 minutes and washed twice in TBS. Chromogenic detection was achieved by applying 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate to the sections for 5 minutes, resulting in a brown precipitate indicating positive antigen expression. Slides were counterstained with Harris hematoxylin, air-dried, and permanently mounted using DPX (Dibutylphthalate Polystyrene Xylene) for visualization under a light microscope. The scoring for IHC was done using the following labelling index,\u003c/p\u003e \u003cp\u003e% of IHC\u0026thinsp;+\u0026thinsp;Labelled cells\u003c/p\u003e\n\u003ch3\u003e0 = 0%\u003c/h3\u003e\n\u003cp\u003e1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;30%\u003c/p\u003e \u003cp\u003e2\u0026thinsp;=\u0026thinsp;30\u0026ndash;60%\u003c/p\u003e \u003cp\u003e3\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;60% [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eSlides were independently reviewed by two pathologists to assess the expression levels of CD34, CD68, MMP9, and VEGF.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAs presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, no VEGF expression was detected, and less than 10% positivity was observed for CD34, and CD68 in the case of Orofacial Granulomatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). For Inflammatory Fibroepithelial Hyperplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), more than 40% positivity was noted for CD34 and VEGF among inflammatory cells, while less than 10% of cells were positive for CD68, and MMP9 exhibited non-specific staining. In Oral Focal Mucinosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), less than 10% of cells showed positivity for VEGF, approximately 5% for CD34, and no positivity was observed for MMP9 or CD68.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunohistochemical report of the 03 cases\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCase No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMMP9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCD34\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCD68\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e165\u0026thinsp;\u0026minus;\u0026thinsp;22 Orofacial granulomatosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;10% focal positivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;30% positivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10% positivity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e181\u0026thinsp;\u0026minus;\u0026thinsp;21 Inflammatory fibroepithelial hyperplasia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;40% positivity in inflammatory cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNon- specific\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;40% positivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10% positivity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e225-21Oral Focal mucinosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;15% focal positivity in inflammatory cell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5% positivity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe expression pattern of VEGF,CD 34, MMP 9, and CD 68 seen in our cases shows the complex process and highlights the potential implications. VEGF is a critical signalling protein involved in the formation of new blood vessels and the regulation of vascular permeability [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the context of orofacial granulomatosis (OFG), inflammatory fibroepithelial hyperplasia, and oral focal mucinosis (OFM), VEGF have been considered in the pathogenesis and progression of these conditions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. OFG is characterized by chronic inflammation, which often leads to tissue swelling and the formation of granulomas. VEGF may contribute to vascularization of these granulomas, to support the immune response within the granuloma VEGF increases vascular permeability, leading to leakage of fluid from blood vessels into the surrounding tissues [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The non expression pattern of VEGF in our case may indicate tha t this case could indicate a long standing granuloma. A negative or low expression of VEGF might indicate that the inflammatory response in OFG is not associated with significant new blood vessel formation. This could potentially result in less pronounced tissue swelling or less severe edema compared to cases where VEGF is highly expressed [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Granulomas in OFG are often sustained by an active inflammatory response and the formation of new blood vessels. If VEGF is low or absent, the granulomas might be less vascularized, possibly leading to a different composition or structure of the granulomas. This could influence the chronicity of the granulomas, potentially making them more stable or less prone to changes over time [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. VEGF is also involved in increasing vascular permeability, which contributes to the inflammatory exudate and edema seen in inflamed tissues. A lack of VEGF might result in a different inflammatory profile, with potentially fewer exudative features and a different pattern of immune cell infiltration. This could lead to a less aggressive inflammatory response in OFG, possibly affecting the severity of the disease. The clinical features of OFG, such as lip swelling, facial edema, and oral mucosal changes, could be less pronounced if VEGF expression is low. The degree of tissue swelling and the extent of visible granulomatous changes might be reduced in cases with negative VEGF expression. Patients with low VEGF might present with milder symptoms or a different distribution of lesions compared to those with high VEGF expression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].The absence of VEGF expression could be used as a diagnostic marker to differentiate between different subtypes or severities of OFG. It might also help distinguish OFG from other granulomatous conditions where VEGF is typically upregulated. In cases where VEGF is not driving the disease process, anti-angiogenic therapies targeting VEGF might not be effective. Instead, other therapeutic strategies targeting different aspects of the inflammatory response or granuloma formation might be more appropriate [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Inflammatory fibroepithelial hyperplasia involves the proliferation of fibrous connective tissue and overlying epithelium in response to chronic irritation or inflammation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. VEGF promotes angiogenesis in these hyperplastic tissues, supporting their growth and maintenance. The increased expression of VEGF in these lesions can lead to the development of a dense network of new blood vessels, which is often observed in hyperplastic tissues. These vascular changes can contribute to the persistence and expansion of the lesion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In inflammatory fibroepithelial hyperplasia, \u0026lt;\u0026thinsp;40% positivity for Vascular Endothelial Growth Factor (VEGF) among inflammatory cells indicates that less than 40% of the inflammatory cells in the tissue express VEGF. \u0026lt;40% positivity suggests that VEGF expression is present but not extensive. This implies that angiogenesis, or the formation of new blood vessels, is occurring at a moderate level in the affected tissue. The limited VEGF expression indicates that while there is some vascular proliferation, it is not the predominant feature. The tissue may have a moderate degree of new blood vessel formation compared to conditions with high VEGF expression and that VEGF is contributing to the inflammatory process but not overwhelmingly. The moderate VEGF expression might reflect a balanced role in supporting tissue repair and inflammation, rather than driving excessive angiogenesis. While\u0026thinsp;\u0026lt;\u0026thinsp;40% positivity indicates moderate VEGF expression, it helps to understand the extent of angiogenic activity in the lesion. This level of VEGF expression can provide insights into the balance between inflammation and tissue repair processes. Treatment strategies might need to address both the inflammatory and hyperplastic components of the lesion. OFM is characterized by the localized accumulation of mucin within the connective tissue. VEGF may play a role in the vascularization of these mucinous nodules, ensuring an adequate blood supply to the affected area. Although OFM is a rare condition, its reactive nature to local stimuli might involve the upregulation of VEGF, which facilitates angiogenesis and supports the mucinous changes within the oral mucosa. VEGF plays a crucial role in the pathogenesis of inflammatory fibroepithelial hyperplasia, and have limited expression in long standing granulomas and in OFM. Understanding VEGF's involvement in these conditions can provide insights into potential diagnostic markers and therapeutic targets. MMP 9 is an enzyme that plays a significant role in the remodeling of the extracellular matrix (ECM) by degrading type IV collagen, a major component of the basement membrane. MMP-9 is involved in various physiological and pathological processes, including inflammation, tissue repair, and tumor progression.\u003c/p\u003e \u003cp\u003eIn OFG, chronic inflammation leads to the formation of granulomas, which are aggregates of immune cells attempting to contain the inflammation. MMP-9 is upregulated in response to inflammatory cytokines and is involved in the remodeling of the ECM surrounding granulomas [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This remodeling is essential for granuloma maintenance and expansion, as MMP-9 facilitates the migration of immune cells to the site of inflammation by breaking down the ECM. MMP-9 contributes to the degradation of ECM components, leading to tissue remodeling. This process is often associated with the swelling and fibrosis seen in OFG. The increased activity of MMP-9 can result in the breakdown of the basement membrane, allowing immune cells to infiltrate the tissue and exacerbate inflammation. The non expression of MMP9 further reiterates that our case is a long standing lesion. In orofacial granulomatosis (OFG), \u0026lt;\u0026thinsp;10% focal positivity for Matrix Metalloproteinase-9 (MMP-9) means that MMP-9 expression is detected in less than 10% of the examined tissue areas. This level of expression provides specific insights into the role of MMP-9 in the disease. Here's an analysis\u0026thinsp;\u0026lt;\u0026thinsp;10% focal positivity indicates that MMP-9 is expressed in only a small portion of the tissue. This suggests that its role in the pathology of OFG is limited or localized rather than widespread. The small areas showing MMP-9 positivity may reflect specific regions where extracellular matrix remodeling is occurring, but overall, MMP-9 may not be a major factor in the disease process. Granulomas in OFG are characterized by a collection of macrophages and other immune cells. Limited MMP-9 expression suggests that the remodeling of extracellular matrix components is not a prominent feature of the granulomas. Low MMP-9 positivity implies that matrix degradation or remodeling might not be a significant part of the inflammatory response in OFG, or that it occurs only in specific, localized areas. \u0026lt;10% positivity means MMP-9 is not a primary marker for OFG. Its limited expression suggests that other factors or pathways might be more relevant to the disease pathology. The low level of MMP-9 expression helps differentiate OFG from conditions with higher MMP-9 positivity, where matrix remodeling and tissue damage might be more prominent [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Since MMP-9 is not highly expressed, targeting MMP-9 may not be particularly effective for treating OFG. The focus of treatment might need to address other aspects of the inflammatory response or granuloma formation. Monitoring MMP-9 levels could be useful for assessing localized matrix remodeling but may not provide comprehensive insights into disease progression or response to treatment. Other biomarkers or clinical parameters should be considered for a complete evaluation. The low focal positivity of MMP-9 suggests that its role in OFG might be less significant compared to other conditions with more pronounced MMP-9 activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Research could focus on understanding why MMP-9 is minimally expressed and identify other key factors driving the disease. In inflammatory fibroepithelial hyperplasia, there is a reactive proliferation of fibrous tissue and epithelium in response to chronic irritation. MMP-9 plays a key role in this process by degrading the ECM, which allows for the expansion of the hyperplastic tissue. This degradation facilitates the migration and proliferation of fibroblasts and epithelial cells, contributing to the growth of the hyperplastic lesion. MMP-9 is often upregulated in chronic inflammatory conditions and is produced by various cells, including macrophages, neutrophils, and fibroblasts. In inflammatory fibroepithelial hyperplasia, the persistent inflammation and tissue irritation likely lead to increased MMP-9 expression, which further drives tissue remodeling and lesion progression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the context of inflammatory fibroepithelial hyperplasia, nonspecific expression of Matrix Metalloproteinase-9 (MMP-9) means that the enzyme is present, but its expression does not correlate strongly with specific pathological features of the condition. MMP-9 is involved in the breakdown of extracellular matrix components, which plays a role in tissue remodeling, inflammation, and repair. Nonspecific expression suggests that MMP-9 is involved in general tissue remodeling and matrix turnover rather than a targeted pathological process. Also that it may contribute to the overall remodeling of fibrous and epithelial tissues but is not specifically driving the hyperplastic changes.MMP-9 is often upregulated in various inflammatory conditions due to its role in tissue damage and repair. Nonspecific expression means that while MMP-9 is present, it is not uniquely associated with the inflammation in this specific condition. The enzyme's expression may reflect a broad inflammatory response rather than a specific contributor to the inflammatory or hyperplastic process in fibroepithelial hyperplasia [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Oral focal mucinosis is characterized by the localized accumulation of mucin within the connective tissue. MMP-9 may be involved in the ECM remodeling that occurs in response to this mucinous change. By degrading ECM components, MMP-9 could facilitate the formation and expansion of mucinous nodules [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In oral focal mucinosis, a negative result for Matrix Metalloproteinase-9 (MMP-9) indicates that MMP-9 is not expressed or detected in the tissue. MMP-9 is an enzyme involved in the breakdown of extracellular matrix components, and its expression is often associated with tissue remodeling, inflammation, and certain pathological conditions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Non expression of MMP-9 suggests that there is little to no breakdown of extracellular matrix (ECM) components by this enzyme in the tissue. This implies that the pathological changes in oral focal mucinosis are not associated with significant ECM remodeling mediated by MMP-9. Since MMP-9 is involved in ECM degradation, its absence might indicate that mucin accumulation in oral focal mucinosis is not associated with extensive tissue remodeling or degradation of ECM proteins. The absence of MMP-9 expression highlights that inflammation in oral focal mucinosis may not involve significant ECM degradation or be driven by MMP-9-mediated processes. CD34 is a cell surface glycoprotein that functions as a marker of hematopoietic stem cells, endothelial cells, and progenitor cells involved in blood vessel formation. It is widely used as a marker for identifying and quantifying blood vessels, particularly in the context of angiogenesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. CD34- positive blood vessels are often observed in areas of active inflammation and granuloma formation in OFG. The presence of CD34-positive vessels can be indicative of the extent of vascular involvement and the degree of angiogenesis associated with the inflammatory process in OFG as observed in our case. In orofacial granulomatosis (OFG), \u0026gt;\u0026thinsp;30% positivity for CD34 indicates that more than 30% of the examined tissue areas exhibit significant expression of CD34. CD34 is a cell surface glycoprotein commonly used as a marker for endothelial cells and hematopoietic progenitor cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. CD34 positivity is often associated with endothelial cells and neovascularization. In OFG, \u0026gt;\u0026thinsp;30% positivity indicates significant vascular proliferation, suggesting that the granulomatous lesions are highly vascularized. This increased vascularization can contribute to tissue swelling and redness, typical of OFG [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The high percentage of CD34- positive areas reflects a high density of blood vessels in the affected tissue, which may be involved in the inflammatory response and the overall pathology of OFG. Granulomas in OFG may exhibit increased angiogenesis as part of the inflammatory response. CD34 positivity highlights that new blood vessel formation is a prominent feature of the granulomas. The presence of numerous blood vessels can contribute to the observed symptoms of OFG, such as swelling, pain, and erythema. High CD34 positivity can be indicative of active disease with significant inflammatory and vascular components. It helps in distinguishing OFG from other conditions with less pronounced vascular changes. Tracking CD34 positivity can be useful for monitoring disease activity and assessing responses to treatments aimed at reducing inflammation and angiogenesis. The high vascularization observed with \u0026gt;\u0026thinsp;30% CD34 positivity suggests that therapies targeting angiogenesis might be beneficial. Treatments that reduce blood vessel formation could help alleviate symptoms and potentially improve clinical outcomes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInflammatory fibroepithelial hyperplasia involves the proliferation of fibrous tissue and epithelium in response to chronic irritation. This process is often accompanied by increased blood vessel formation to support the growing tissue. CD34 is a key marker for identifying these new blood vessels. The degree of CD34 expression can provide insights into the level of angiogenesis occurring within the hyperplastic tissues. As part of the tissue's response to chronic irritation, CD34- positive endothelial progenitor cells may contribute to the repair and regeneration processes by promoting new blood vessel formation. This vascular support is crucial for the maintenance and growth of the hyperplastic lesion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Oral focal mucinosis is characterized by the localized accumulation of mucin within the connective tissue. The formation of these mucinous nodules is often accompanied by the development of new blood vessels to support the tissue changes. Limited CD34 expression in our case can be used to assess the extent of vascularization within these nodules. Given that OFM is a reactive condition, the presence of CD34- positive blood vessels may reflect the tissue's response to local stimuli. CD68 is a glycoprotein primarily expressed on the surface of monocytes and macrophages, making it an important marker for identifying and studying these immune cells. CD68 is widely used as a marker to identify these macrophages within granulomatous lesions. The presence of CD68- positive macrophages in granulomas indicates their role in containing the inflammatory stimulus and in orchestrating the immune response. In OFG, macrophages play a critical role in sustaining the chronic inflammatory response by secreting pro-inflammatory cytokines and chemokines. CD68 expression is a marker of macrophage activation and can be used to assess the extent of macrophage involvement in the disease process. High levels of CD68- positive cells are indicative of active inflammation and granulomatous tissue formation [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn orofacial granulomatosis (OFG), \u0026gt;\u0026thinsp;10% positivity for CD68 suggests that more than 10% of the examined tissue areas exhibit significant expression of CD68. CD68 is a marker commonly associated with macrophages, which are key players in inflammation and immune responses. CD68 positivity indicates the presence of macrophages in the tissue. \u0026gt;10% positivity suggests a notable infiltration of macrophages within the granulomas of OFG, highlighting the role of these cells in the inflammation. Macrophages are integral to granuloma formation. Their significant presence supports the diagnosis of OFG, as these cells are crucial in the formation and maintenance of granulomatous inflammation. High levels of CD68 positivity point to an active inflammatory response in OFG. Macrophages play a central role in chronic inflammation and tissue response to irritants or foreign substances. The presence of macrophages suggests ongoing immune activation and response, which is a hallmark of granulomatous conditions.\u0026gt;10% positivity for CD68 is indicative of significant macrophage activity and can help confirm the presence of OFG. It supports the diagnosis by demonstrating the extent of inflammatory involvement. This level of CD68 positivity helps differentiate OFG from other oral lesions where macrophage infiltration might be less pronounced [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The significant presence of macrophages suggests that treatment strategies might need to focus on modulating the inflammatory response. Therapies that target macrophage activity or the inflammatory pathways they are involved in could be beneficial. In inflammatory fibroepithelial hyperplasia, chronic irritation leads to the proliferation of fibrous tissue. Macrophages, marked by CD68, are often present in these lesions as part of the chronic inflammatory response. They may contribute to tissue remodeling by releasing enzymes and growth factors that influence fibroblast activity and collagen production, leading to the thickening of the fibroepithelial tissue. Macrophages are also involved in tissue repair processes, where they help clear debris, secrete factors that promote healing, and modulate inflammation. The presence of CD68- positive macrophages in hyperplastic tissue could indicate ongoing tissue remodeling and repair in response to chronic irritation or inflammation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In inflammatory fibroepithelial hyperplasia, \u0026gt;\u0026thinsp;10% positive for CD68 indicates that more than 10% of the examined tissue areas exhibit significant expression of CD68. \u0026gt;10% positivity for CD68 suggests that a substantial proportion of the inflammatory cells in the tissue are macrophages. The presence of macrophages is indicative of ongoing inflammation. These cells are involved in the inflammatory response, tissue repair, and the regulation of immune responses. Although OFM is a relatively benign condition, macrophages may still play a role in the reactive processes. CD68- positive macrophages could be involved in clearing cellular debris, responding to local stimuli, and possibly modulating the accumulation of mucin within the tissue. Even though OFM is not primarily an inflammatory condition, the presence of CD68-positive macrophages might be observed in cases where there is a mild or localized inflammatory response associated with the mucinous nodules. These macrophages could be involved in maintaining tissue homeostasis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In our case negative expression of CD68 suggests that macrophages, or cells with macrophage-like properties, are not present in the tissue or are present in such low numbers that they are below the detection threshold and so there is minimal or no macrophage-driven inflammation in oral focal mucinosis. The negative CD68 result supports the idea that the condition is primarily due to mucin deposition rather than an inflammatory response involving macrophages\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBy analyzing these markers, clinicians and researchers can gain a better understanding of the underlying mechanisms driving these oral pathologies, potentially leading to more effective management and treatment strategies.\u003c/p\u003e\n\u003ch3\u003eFuture scope/ clinical significance\u003c/h3\u003e\n\u003cp\u003eThis study highlights the differential expression of VEGF, MMP-9, CD34, and CD68 across orofacial granulomatosis (OFG), inflammatory fibroepithelial hyperplasia, and oral focal mucinosis (OFM). Future research could validate these markers' diagnostic and prognostic utility in larger cohorts. Identifying thresholds for biomarker positivity may help refine diagnostic criteria, enabling earlier and more accurate differentiation between these conditions. Further investigation into VEGF and MMP-9 pathways may reveal novel therapeutic targets, particularly in cases with prominent angiogenesis and extracellular matrix remodeling.For conditions with low VEGF and MMP-9 expression, studies could explore alternative mechanisms driving pathogenesis and identify more suitable therapeutic approaches.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOFG- Orofacial Granulomatosis\u003c/p\u003e\n\u003cp\u003eOFM- Oral Focal Mucinosis\u003c/p\u003e\n\u003cp\u003eVEGF- Vascular Endothelial Growth Factor\u003c/p\u003e\n\u003cp\u003eMMP- Metallic metalloproteinase\u003c/p\u003e\n\u003cp\u003eESR- Erythrocyte Sedimentation Rate\u003c/p\u003e\n\u003cp\u003eQFT- . Quantiferon TB gold Test\u003c/p\u003e\n\u003cp\u003eACE- Angiotensin- Converting Enzyme\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIOPA- Intraoral Periapical Radiograph\u003c/p\u003e\n\u003cp\u003eLA- Local Anaesthesia\u003c/p\u003e\n\u003cp\u003eIHC- Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eTBS- Tris Buffer Saline\u003c/p\u003e\n\u003cp\u003eDAB- \u0026nbsp;3,30-diaminobenzidine tetrahydrochloride\u003c/p\u003e\n\u003cp\u003eDPX- Dibutylphthalate Polystyrene Xylene\u003c/p\u003e\n\u003cp\u003eECM- Extra Cellular Matrix\u003c/p\u003e\n\u003cp\u003eMMP- Matrix Metalloprotease\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval and informed consent to participate was obtained from all of the participants in the study by Saveetha Dental College and Hospitals Institutional Ethics Committee (SDC-IHEC), Registered under Government of India, (ECR/1452/Inst/TN/2024). The study adhered to the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePR, RD, JND, CS, and AR contributed to conceptualization; PR, RD, AR contributed to methodology; RD, JND, CS, AR contributed to validation; RD, PR, JND, CS, NU, and MRM contributed to formal analysis; RD, PR and AR contributed in formal analysis, investigation and resources; GS and AR were involved in data curation; RD, PR and AR were involved in writing- original draft preparation, review and editing; RD, PR, JND, CS and AR involved in visualization; RD and PR involved in supervision. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge Dr. N.M. Veeraiyan, Chancellor, and Dr. Deepak Nallaswamy SIMATS University for providing the extended essential facilities for the research work. The authors acknowledge Dr. Nisha Jaishree, MDS, Oral Pathology, SIMATS for helping in conducting the Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Oral Pathology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, Tamilnadu- 600077\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eDepartment of Periodontology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India- 600077\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eDepartment of Oral and Maxillofacial Surgery, EHS, Fujairah Specialized Dental Center and Hospital, Fujairah, UAE\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSuresh R, Ramadoss R, Krishnasamy N, Selvam SP, Sundar S, K H. 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J Cutan Pathol. 2021;48:863\u0026ndash;9. https://doi.org/10.1111/cup.13970.\u003c/li\u003e\n\u003cli\u003eNiklander S, Bordagaray MJ, Fern\u0026aacute;ndez A, Hern\u0026aacute;ndez M. Vascular Endothelial Growth Factor: A Translational View in Oral Non-Communicable Diseases. Biomolecules. 2021;11:85. https://doi.org/10.3390/biom11010085.\u003c/li\u003e\n\u003cli\u003eShukla C, Singla S, Mali S, Niranjan B, Shashikiran ND, Dubey A. A Rare Gingival Lesion in Children: Fibroepithelial Hyperplasia. International Journal of Clinical Pediatric Dentistry. 2022;15:468\u0026ndash;71. https://doi.org/10.5005/jp-journals-10005-2412.\u003c/li\u003e\n\u003cli\u003eLee H-W, Xu Y, He L, Choi W, Gonzalez D, Jin S-W, et al. Role of Venous Endothelial Cells in Developmental and Pathologic Angiogenesis. Circulation. 2021;144:1308\u0026ndash;22. https://doi.org/10.1161/CIRCULATIONAHA.121.054071.\u003c/li\u003e\n\u003cli\u003eAmaral EP, Vinhaes CL, Oliveira-de-Souza D, Nogueira B, Akrami KM, Andrade BB. 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Front Aging Neurosci. 2021;13:711784. https://doi.org/10.3389/fnagi.2021.711784.\u003c/li\u003e\n\u003cli\u003eGaldiero MR, Maio F, Arcoleo F, Boni E, Bonzano L, Brussino L, et al. Orofacial granulomatosis: Clinical and therapeutic features in an Italian cohort and review of the literature. Allergy. 2021;76:2189\u0026ndash;200. https://doi.org/10.1111/all.14799.\u003c/li\u003e\n\u003cli\u003eShimizu Y. Mechanism underlying vascular remodeling in relation to circulating CD34-positive cells among older Japanese men. Sci Rep. 2022;12:21823. https://doi.org/10.1038/s41598-022-26089-y.\u003c/li\u003e\n\u003cli\u003eHassanpour M, Salybekov AA, Kobayashi S, Asahara T. CD34 positive cells as endothelial progenitor cells in biology and medicine. Front Cell Dev Biol. 2023;11:1128134. https://doi.org/10.3389/fcell.2023.1128134.\u003c/li\u003e\n\u003cli\u003eLabrador AJP, Valdez LHM, Marin NRG, Ibazetta KAR, Chac\u0026oacute;n JAL, Fernandez AJV, et al. Oral granulomatosis with polyangiitis a systematic review. Clinical \u0026amp; Exp Dental Res. 2023;9:100\u0026ndash;11. https://doi.org/10.1002/cre2.706.\u003c/li\u003e\n\u003cli\u003eSekar K, Ramanathan A, Khalid R, Mun KS, Valliappan V, Ismail SM. An unusual case of multiple primary tumours involving the long bone and oral cavity. Oral Maxillofac Surg. 2025;29:62. https://doi.org/10.1007/s10006-025-01356-0.\u003c/li\u003e\n\u003cli\u003eJiang C, Wang W, Chen Y-L, Chen J-H, Zhang Z-W, Li J, et al. Macrophage polarization and macrophage-related factor expression in hypertrophy of the ligamentum flavum. Eur Spine J. 2024. https://doi.org/10.1007/s00586-024-08513-1.\u003c/li\u003e\n\u003cli\u003eLehman JS, Sokumbi O, Peters MS, Bridges AG, Comfere NI, Gibson LE, et al. Histopathologic features of noninfectious granulomatous disorders involving the skin. Human Pathology. 2020;103:127\u0026ndash;45. https://doi.org/10.1016/j.humpath.2020.05.008.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Case Series, Vascular Endothelial Growth Factor (VEGF), Matrix metalloproteinase-9 (MMP-9), Orofacial granulomatosis (OFG), CD38, Fibroepithelial hyperplasia, mucinosis, vascularization, biomarkers","lastPublishedDoi":"10.21203/rs.3.rs-8891946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8891946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eOrofacial granulomatosis (OFG), inflammatory fibroepithelial hyperplasia (IFH), and oral focal mucinosis (OFM) share characteristics of chronic inflammation and tissue remodeling in response to local irritation or immune dysregulation within the oral cavity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study investigates the expression of VEGF, MMP-9, CD68, and CD34 in OFG, IFH, and OFM, aiming to identify potential overlapping etiological factors, inflammatory pathways, and clinical patterns. By examining these molecular markers, we seek to uncover possible commonalities in pathogenesis that may guide diagnosis, inform treatment approaches, and improve management strategies for these conditions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCase analyses included histopathological and immunohistochemical evaluations. In OFG, CD34, MMP-9, and CD68 expression were under 10%, with no VEGF detected. In contrast, IFH showed over 40% positivity for CD34 and VEGF among inflammatory cells, low CD68 (\u0026lt;\u0026thinsp;10%), and non-specific MMP-9 staining. OFM displayed less than 10% VEGF expression, minimal CD34 positivity (5%), and absent MMP-9 and CD68.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest distinct biomarker expression patterns that reflect vascularity and inflammatory profiles, which could aid in diagnosis and management. Specifically, VEGF expression was moderate in IFH, minimal in OFG, and absent in OFM, indicating varying angiogenic activity. The prominent presence of CD34 and CD68 in OFG and IFH highlights immune cell involvement, while minimal MMP-9 suggests limited extracellular matrix remodeling. Overall, these results offer insights into the inflammatory and vascular characteristics of these oral conditions.\u003c/p\u003e","manuscriptTitle":"Expression pattern of VEGF, MMP 9,CD34 and CD68 in Orofacial Granulomatosis, Inflammatory Fibroepithelial Hyperplasia, and Oral Focal Mucinosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 19:59:56","doi":"10.21203/rs.3.rs-8891946/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-29T10:17:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-29T00:05:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T19:25:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T17:05:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2628778505355837380714753745693225918","date":"2026-04-24T13:33:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54184054858330644944380643049174393147","date":"2026-04-23T07:55:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338162149506747930015827635809067527724","date":"2026-04-23T07:48:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-23T07:43:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-06T10:25:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-23T14:16:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T06:56:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2026-02-23T06:50:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c45a20b-604c-4b08-94cd-e243e63c1608","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T12:08:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 19:59:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8891946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8891946","identity":"rs-8891946","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}