Correlation between Vascularity and Advancing Histological Grades of Oral Submucous Fibrosis with a Plausible Role in Malignisation: Systematic review of a persisting matter of conflict.

Objectives: This study aimed to quantify the vascularity in histological grades of oral submucous fibrosis (OSMF) and to determine if there is any connection between vasculogenesis and malignisation. Recent studies show no significant change in vascularity as the stage advances as opposed to the conventional concept. Methods: A comprehensive database search until December 2022 was conducted for published articles on vascularity in OSMF following preferred reporting items for systematic reviews and meta-analyses guidelines. Results: A total of 98 articles were screened of which 13 were included for systematic evaluation. The study included 607 cases, with a definite predilection for the male gender. Of the 13 studies, 11 evaluated mean vascular density. In more than half of the studies, the vascularity decreased as the stage advanced. Similar results were obtained for endothelial cells/µm2, mean vascular area percentage and mean vascular area. Conclusion: The present review supports the prevailing concept that vascularity decreases with the advancement of the OSMF stage. This denies the systemic absorption of carcinogens into the circulation with resultant longer exposure of compromised epithelium and malignisation.

HLA-DQB1 Allele Polymorphism Associated with Oral Submucous Fibrosis in Hunan, China.

Methods: 44 OSF patients and 44 healthy volunteers were included in this study. To detect the expression frequency of HLA-DQB1 alleles in the two groups and analyze significant allelic subtypes and their relative risk, polymerase chain reaction (PCR) sequence-specific primers were used. Subsequently, based on the identification of differential genes, we compare the gene expression levels of OSF patients and healthy volunteers expressing differential genes by real-time quantitative PCR. Results: The expression frequency of the HLA-DQB1 ∗05 : 02 allele in the OSF group (36.4%) was significantly higher than in the controls (13.6%), and exposure to the HLA-DQB1 ∗05 : 02 allele was strongly related to OSF (OR (95% CI) = 3.619 (1.257,10.421), Wald χ2 = 5.681, P=0.017). However, there were no significant differences in the allele expression frequencies of DQB1 ∗02 : 01, DQB1 ∗03 : 03, DQB1 ∗05 : 01, DQB1 ∗05 : 03, DQB1 ∗06 : 02, DQB1 ∗06 : 03, and DQB1 ∗06 : 04 in the OSF group compared with the controls (all P > 0.05). Furthermore, the relative expression level of the HLA-DQB1 ∗05 : 02 allele in the OSF group (3.98 ± 3.50) was significantly higher than in controls (0.70 ± 0.41). Conclusions: There are differences in the HLA-DQB1 allele polymorphisms between the healthy population and patients with oral submucosal fibrosis. Preliminarily, it is suggested that the HLA-DQB1 ∗05 : 02 allele, which has a strong correlation with OSF and great differential expression between patients with OSF and controls, might be a susceptibility gene for OSF in Hunan.

DPSCs regulate epithelial-T cell interactions in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion characterized by fibrous tissue deposition, the incidence of which correlates positively with the frequency of betel nut chewing. Prolonged betel nut chewing can damage the integrity of the oral mucosal epithelium, leading to chronic inflammation and local immunological derangement. However, currently, the underlying cellular events driving fibrogenesis and dysfunction are incompletely understood, such that OSF has few treatment options with limited therapeutic effectiveness. Dental pulp stem cells (DPSCs) have been recognized for their anti-inflammatory and anti-fibrosis capabilities, making them promising candidates to treat a range of immune, inflammatory, and fibrotic diseases. However, the application of DPSCs in OSF is inconclusive. Therefore, this study aimed to explore the pathogenic mechanism of OSF and, based on this, to explore new treatment options. METHODS: A human cell atlas of oral mucosal tissues was compiled using single-cell RNA sequencing to delve into the underlying mechanisms. Epithelial cells were reclustered to observe the heterogeneity of OSF epithelial cells and their communication with immune cells. The results were validated in vitro, in clinicopathological sections, and in animal models. In vivo, the therapeutic effect and mechanism of DPSCs were characterized by histological staining, immunohistochemical staining, scanning electron microscopy, and atomic force microscopy. RESULTS: A unique epithelial cell population, Epi1.2, with proinflammatory and profibrotic functions, was predominantly found in OSF. Epi1.2 cells also induced the fibrotic process in fibroblasts by interacting with T cells through receptor-ligand crosstalk between macrophage migration inhibitory factor (MIF)-CD74 and C-X-C motif chemokine receptor 4 (CXCR4). Furthermore, we developed OSF animal models and simulated the clinical local injection process in the rat buccal mucosa using DPSCs to assess their therapeutic impact and mechanism. In the OSF rat model, DPSCs demonstrated superior therapeutic effects compared with the positive control (glucocorticoids), including reducing collagen deposition and promoting blood vessel regeneration. DPSCs mediated immune homeostasis primarily by regulating the numbers of KRT19 + MIF + epithelial cells and via epithelial-stromal crosstalk. CONCLUSIONS: Given the current ambiguity surrounding the cause of OSF and the limited treatment options available, our study reveals that epithelial cells and their crosstalk with T cells play an important role in the mechanism of OSF and suggests the therapeutic promise of DPSCs.

Evaluation of stromal myofibroblasts in oral submucous fibrosis and its malignant transformation: An immunohistochemical study.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous lesion, with oral squamous cell carcinoma (OSCC) being the most prevalent malignancy affecting the oral mucosa. The malignant transformation of OSF into OSCC is estimated to occur in 7-13% of cases. Myofibroblasts (MFs) play pivotal roles in both physiological and pathological processes, such as wound healing and tumorigenesis, respectively. This study aimed to explore the involvement of MFs in the progression of OSF and its malignant transformation. MATERIALS AND METHODS: In total, 94 formalin-fixed paraffin-embedded tissue blocks were collected, including normal oral mucosa (NOM; n = 10), early-moderate OSF (EMOSF; n = 29), advanced OSF (AOSF; n = 29), paracancerous OSF (POSF; n = 21), and OSCC (n = 5) samples. Alpha-smooth muscle actin was used for the immunohistochemical identification of MFs. RESULTS: NOM exhibited infrequent expression of MFs. A higher staining index of MFs was found in AOSF, followed by EMOSF and NOM. Additionally, a significant increase in the staining index of MFs was found from EMOSF to POSF and OSCC. The staining index of MFs in NOM, EMOSF, AOSF, POSF, and OSCC was 0.14 ± 0.2, 1.69 ± 1.4, 2.47 ± 1.2, 3.57 ± 2.6, and 8.86 ± 1.4, respectively. All results were statistically significant (P < 0.05). CONCLUSIONS: The expression of MFs exhibited a gradual increase as the disease progressed from mild to malignant transformation, indicating the contributory role of MFs in the fibrogenesis and potential tumorigenesis associated with OSF.

Salivary and serum levels of lactate dehydrogenase in oral submucous fibrosis: A meta-analysis.

BACKGROUND: The occurrence of oral submucous fibrosis (OSF) is often accompanied by an increase in lactate dehydrogenase (LDH) levels. In this meta-analysis, we compared the salivary and serum levels of LDH levels between OSF patients and controls. MATERIAL AND METHODS: A comprehensive search was conducted in PubMed, Embase, Web of Science, and Cochrane Library from the establishment of the database to June 2023, and the quality of the studies was checked by the Newcastle-Ottawa Quality Assessment scale. The mean difference (MD) and 95% confidence interval (CI) were calculated using RevMan 5.4 software. RESULTS: A total of 28 studies were retrieved from the database, and we included 5 studies in this meta-analysis. The salivary LDH level of OSF patients was higher than healthy controls (MD: 423.10 pg/L 95%CI: 276.42-569.77 pg/mL, P < .00001), the serum LDH level of OSF patients was also higher than that of healthy controls (MD: 226.20 pg/mL, 95%CI: 147.71-304.69 pg/mL, P < .00001). CONCLUSIONS: This meta-analysis showed that salivary and serum LDH levels were higher in OSF patients than in healthy controls, suggesting that LDH may be a potential biomarker for OSF.

Expression Profile of Circulating Exosomal microRNAs in Leukoplakia, Oral Submucous Fibrosis, and Combined Lesions of Leukoplakia and Oral Submucous Fibrosis.

OBJECTIVES: This study aims to elucidate the expression of circulating exosomal miRNAs miRNA 21, miRNA 184, and miRNA 145 in the studied groups, including patients with (i) leukoplakia; (ii) oral submucous fibrosis; (iii) oral submucous fibrosis with leukoplakia; (iv) oral squamous cell carcinoma; and (v) healthy individuals. STUDY DESIGN: An observational study was conducted among 54 patients who reported to the outpatient department of Saveetha Dental College and Hospitals. The patients were divided into three groups: Group I healthy individuals (n = 18), Group II: case group (leukoplakia, OSMF, and leukoplakia and OSMF) (n = 18), and Group III: OSCC (n = 18). Real-time polymerase chain reaction analysis was carried out to assess the expression profiles of miRNA 21, miRNA 184, and miRNA 145. The statistical analysis was calculated using SPSS software version 23. RESULTS: All three miRNAs showed a statistically significant difference in the one-way ANOVA test between the case group (leukoplakia, OSMF, and leukoplakia and OSMF), healthy group, and OSCC group (p < 0.005). The case group (leukoplakia, OSMF, leukoplakia and OSMF) showed upregulated expression of miRNA 21 and miRNA 184 with threefold change and fourfold change and downregulated expression of miRNA 145 with 1.5-fold change when compared to apparently healthy individuals. CONCLUSION: Plasma circulating exosomal miRNAs miRNA 21, miRNA 145, and miRNA 184 expression could be a novel panel of plasma biomarkers to categorise case group (leukoplakia, OSMF, leukoplakia and OSMF) patients with a high risk of malignant transformation.

Salivary transforming growth factor beta in oral submucous fibrosis: A diagnostic and predictive marker.

CONTEXT: Growth factors and cytokines like transforming growth factor beta (TGF-ß) play a key role in the pathogenesis of oral submucous fibrosis. AIMS: To elucidate the role of Salivary TGF-ß isoforms as a predictive and diagnostic marker for oral submucous fibrosis. SETTINGS AND DESIGN: A total of 30 OSMF and 10 control patients were included in this study, and their clinic-epidemiological data was recorded. METHODOLOGY: The expression of TGF-ß genes-TGF-ß1, TGF-ß2, TGF-ß3-was studied by a real-time polymerase chain reaction in tissue and saliva. Patients were given medicinal intervention for 12 weeks along with jaw-opening exercises. Expression of salivary TGF-ß genes was studied at 12 weeks. STATISTICAL ANALYSIS USED: SPSS software version 20. RESULT: Expression of salivary TGF beta isoforms in OSMF was more than in the control group. There was an increase in salivary TGF-ß1, ß2, ß3 expressions with increasing clinical grades of OSMF and advancing the stage of the disease. Expression of all the TGF beta isoforms was decreased after treatment with statistically significant results. Statistically significant correlations were found between the mean difference of TGF-ß1 and the mean difference between mouth opening and tongue protrusion. CONCLUSION: Salivary TGF-ß isoforms may be used in diagnosis, risk assessment, and screening of the entire population at risk of OSMF after its clinical validation. However, adequate sample size and segmental assessment of the expression of TGF-ß isoforms are needed for further evaluation.

Development of Epigallocatechin 3-gallate-Loaded Hydrogel Nanocomposites for Oral Submucous Fibrosis.

Oral submucous fibrosis (OSF) is a chronic progressive disease associated with increased collagen deposition and TGF-ß1 release. The current therapy and management have been a limited success due to low efficacy and adverse drug reactions. This study aimed to evaluate epigallocatechin 3-gallate (EGCG) encapsulated nanoparticles loaded mucoadhesive hydrogel nanocomposite (HNC) for OSF. Developed HNC formulations were evaluated for their permeation behaviour using in vitro as well as ex vivo studies, followed by evaluation of efficacy and safety by in vivo studies using areca nut extract-induced OSF in rats. The disease condition in OSF-induced rats was assessed by mouth-opening and biochemical markers. The optimized polymeric nanoparticles exhibited the required particle size (162.93 ± 13.81 nm), positive zeta potential (22.50 ± 2.94 mV) with better mucoadhesive strength (0.40 ± 0.002 N), and faster permeation due to interactions of the positively charged surface with the negatively charged buccal mucosal membrane. HNC significantly improved disease conditions by reducing TGF-ß1 and collagen concentration without showing toxicity and reverting the fibroid buccal mucosa to normal. Hence, the optimized formulation can be further tested to develop a clinically alternate therapeutic strategy for OSF.

Aberrantly downregulated FENDRR by arecoline elevates ROS and myofibroblast activation via mitigating the miR-214/MFN2 axis.

Long non-coding RNA FENDRR possesses both anti-fibrotic and anti-cancer properties, but its significance in the development of premalignant oral submucous fibrosis (OSF) remains unclear. Here, we showed that FENDRR was downregulated in OSF specimens and fibrotic buccal mucosal fibroblasts (fBMFs), and overexpression of FENDRR mitigated various myofibroblasts hallmarks, and vice versa. In the course of investigating the mechanism underlying the implication of FENDRR in myofibroblast transdifferentiation, we found that FENDRR can directly bind to miR-214 and exhibit its suppressive effect on myofibroblast activation via titrating miR-214. Moreover, we showed that mitofusin 2 (MFN2), a protein that is crucial to the fusion of mitochondria, was a direct target of miR-214. Our data suggested that FENDRR was positively correlated with MFN2 and MFN2 was required for the inhibitory property of FENDRR pertaining to myofibroblast phenotypes. Additionally, our results showed that the FENDRR/miR-214 axis participated in the arecoline-induced reactive oxygen species (ROS) accumulation and myofibroblast transdifferentiation. Building on these results, we concluded that the aberrant downregulation of FENDRR in OSF may be associated with chronic exposure to arecoline, leading to upregulation of ROS and myofibroblast activation via the miR-214-mediated suppression of MFN2.

Change and pathological significance of glycogen content in oral squamous cell carcinoma and oral submucous fibrosis.

OBJECTIVE: This study aimed to investigate the change and pathological significance of glycogen content in oral squamous cell carcinoma (OSCC) and oral submucous fibrosis (OSF). METHODS AND MATERIALS: 13 normal oral mucosa (NOM), 12 OSF mucosa, and 35 pairs of OSCC tissues and their corresponding adjacent mucosa tissues (AT) were collected from Xiangya Hospital for PAS staining to detect glycogen. Transcriptome sequencing data from OSCC were used to compare glycogen metabolism gene expression differences. Kaplan-Meier method was conducted to estimate Recurrence-free survival (RFS). RESULTS: Glycogen levels were lower in OSF than in NOM and lower in OSCC than in AT. Transcriptome sequencing data analysis showed the expression of most glycogenolysis genes was increased and the expression of glycogen synthesis genes including PPP1R3C and GBE1 was decreased in OSCC tissues. High glycogen level was correlated with poor prognosis in OSCC patients under the background of OSF. CONCLUSION: Glycogen may be used as a potential diagnostic biomolecule for OSF and OSCC, as well as a potential prognostic factor for OSCC in the context of OSF.

Longitudinal outcomes of trismus release with free-flap reconstruction in primary and secondary oral submucous fibrosis-related trismus.

BACKGROUND: Oral submucous fibrosis is a global health concern associated with betel quid use and results in trismus, which can be either primary or secondary in origin. Severe cases often require trismus release with free-flap reconstruction. This study examined longitudinal outcome trends following trismus release and compared the outcomes of patients with primary and secondary oral submucous fibrosis-related trismus. METHODS: We conducted a retrospective cohort study by including patients who underwent trismus release between 2013 and 2022. All procedures were performed by a single surgical team to ensure technique standardisation. We measured the maximum mouth opening, the interincisal distance, perioperatively and 1, 2, 3, 4, 6 and 12 months post-operatively. Data were analysed using generalised estimating equations. RESULTS: A total of 35 patients were included in the study, 17 with primary and 18 with secondary oral submucous fibrosis-related trismus. Initially, patients with primary oral submucous fibrosis-related trismus had greater interincisal distance gains than those with secondary oral submucous fibrosis-related trismus (p = 0.015 and p = 0.025 at 3 and 4 months post-operatively, respectively). However, after 12 months, this initial advantage faded, with comparable interincisal distance improvements in patients with primary and secondary disease, despite the more complex surgical procedures required in secondary cases. CONCLUSION: Surgeons should carefully consider the benefits of trismus release procedures for patients with secondary oral submucous fibrosis-related trismus by recognising the changes in post-operative outcomes.

An enigmatic pathogenetic mechanism of hypoxia inducible factor - 1/2 alpha in the progression of fibrosis of oral submucous fibrosis and its malignant transformation: A systematic review and meta-analysis.

OBJECTIVE: Oral submucous fibrosis is a frequently reported potentially malignant disorder characterized by fibrosis and a malignant transformation rate of 7-30%. The role of hypoxia-inducible factor-1/2α in malignant transformation mechanisms of oral submucous fibrosis remains uncharted territory owing to a scarcity of studies. Thus the present systematic review and meta-analysis aimed to determine the role of hypoxia-inducible factor-1/2α in the progression of fibrosis of oral submucous fibrosis and its malignant transformation. MATERIAL AND METHODS: Using PubMed, Google Scholar, and Cochrane Library databases, full-text articles that investigated hypoxia-inducible factor-1/2α in oral submucous fibrosis were entailed for review. A modified Newcastle-Ottawa scale was employed to evaluate risk of bias in all articles and Review Manager was utilized for meta-analysis. RESULTS: Eighteen and eight qualified articles respectively were included for qualitative and quantitative data synthesis. Progressive upregulation of hypoxia-inducible factor-1/2α in oral submucous fibrosis is associated with fibrosis-induced carcinogenesis. A Random-effects model uncloaked that oral submucous fibrosis cases with significantly increased expression of hypoxia-inducible factor-1α had an increased associated risk of malignant transformation compared with controls (combined odds ratio 523.83, 95% confidence interval 125.74- 2182.28, p < 0.00001). CONCLUSION: The existing evidence substantiates the notion that hypoxia-inducible factor-1/2α, a fundamental pathogenetic mechanism of progression and malignant transformation of oral submucous fibrosis in the background of fibrosis.

Association of Oral Submucous Fibrosis Risk with GSTM1 and GSTT1 Gene Polymorphisms.

OBJECTIVE: To determine the association of GSTM1 and GSTT1 polymorphisms with oral submucous fibrosis (OSF). STUDY DESIGN: A case-control study. Place and Duration of the Study: Department of Human Genetics and Molecular Biology, University of Health Sciences, Lahore and Oral and Maxillofacial Surgery Department, de Montmorency, College of Dentistry/ Punjab Dental Hospital, Lahore, Pakistan, from 1st April 2019 to 31st April 2020. METHODOLOGY: OSF patients were diagnosed with different clinical staging of mouth opening by Vernier caliper with the help of a professional dentist in the Department of Oral and Maxillofacial, de Montmorency, College of Dentistry, Lahore. One hundred and eight blood samples of OSF patients and 108 samples of normal controls were collected. Genomic DNA was obtained from whole-blood extraction. Multiplex PCR amplification using GSTM1, GSTT1, and ß -Globin gene primers was performed. RESULTS: GSTM1 and GSTT1 null genotypes frequencies were found in 43.5% (47/108) and 13.9% (15/108) of controls, whereas 54.6% (59/108) and 25.9% (28/108) of OSF patients, respectively. OSF patients had a greater frequency rate of GSTM1 and GSTT1 null genotypes than controls [OR 1.56, 95% CI 0.91-2.67 (p=0.13)] and [OR 2.17, 95% CI 1.08-4.34 (p=0.04)], respectively. The GSTT1 genotype was found statistically significant with OSF (p=0.05), and risk was also determined. The cumulative effect of null genotypes of GSTM1/GSTT1 did not show any association with the controls and in OSF patients. Proportions of active and null alleles of the patient group were; 86.1%/13.9%; and in control, it was 92.6%/7.4% (OR = 2.01; CI: 0.82-4.97; p=0.18), respectively. CONCLUSION: The study determined a statistically significant association of GSTT1 gene polymorphism with OSF. KEY WORDS: Oral submucous fibrosis, GSTM1, GSTT1, Gene polymorphisms, Genetic risk.

Interventions for managing oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a chronic disease of the oral cavity that causes progressive constriction of the cheeks and mouth accompanied by severe pain and reduced mouth opening. OSF has a significant impact on eating and swallowing, affecting quality of life. There is an increased risk of oral malignancy in people with OSF. The main risk factor for OSF is areca nut chewing, and the mainstay of treatment has been behavioural interventions to support habit cessation. This review is an update of a version last published in 2008. OBJECTIVES: To evaluate the benefits and harms of interventions for the management of oral submucous fibrosis. SEARCH METHODS: We used standard, extensive Cochrane search methods. The latest search date was 5 September 2022. SELECTION CRITERIA: We considered randomised controlled trials (RCTs) of adults with a biopsy-confirmed diagnosis of OSF treated with systemic, locally delivered or topical drugs at any dosage, duration or delivery method compared against placebo or each other. We considered surgical procedures compared against other treatments or no active intervention. We also considered other interventions such as physiotherapy, ultrasound or alternative therapies. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. Our primary outcomes were 1. participant-reported resumption of normal eating, chewing and speech; 2. change or improvement in maximal mouth opening (interincisal distance); 3. improvement in range of jaw movement; 4. change in severity of oral/mucosal burning pain/sensation; 5. ADVERSE EFFECTS: Our secondary outcomes were 6. quality of life; 7. postoperative discomfort or pain as a result of the intervention; 8. participant satisfaction; 9. hospital admission; 10. direct costs of medication, hospital bed days and any associated inpatient costs for the surgical interventions. We used GRADE to assess certainty of evidence for each outcome. MAIN RESULTS: We included 30 RCTs (2176 participants) in this updated review. We assessed one study at low risk of bias, five studies at unclear risk of bias and 24 studies at high risk of bias. We found diverse interventions, which we categorised according to putative mechanism of action. We present below our main findings for the comparison 'any intervention compared with placebo or no active treatment' (though most trials included habit cessation for all participants). Results for head-to-head comparisons of active interventions are presented in full in the main review. Any intervention versus placebo or no active treatment Participant-reported resumption of normal eating, chewing and speech No studies reported this outcome. Interincisal distance Antioxidants may increase mouth opening (indicated by interincisal distance (mm)) when measured at less than three months (mean difference (MD) 3.11 mm, 95% confidence interval (CI) 0.46 to 5.77; 2 studies, 520 participants; low-certainty evidence), and probably increase mouth opening slightly at three to six months (MD 8.83 mm, 95% CI 8.22 to 9.45; 3 studies, 620 participants; moderate-certainty evidence). Antioxidants may make no difference to interincisal distance at six-month follow-up or greater (MD -1.41 mm, 95% CI -5.74 to 2.92; 1 study, 90 participants; low-certainty evidence). Pentoxifylline may increase mouth opening slightly (MD 1.80 mm, 95% CI 1.02 to 2.58; 1 study, 106 participants; low-certainty evidence). However, it should be noted that these results are all less than 10 mm, which could be considered the minimal change that is meaningful to someone with oral submucous fibrosis. The evidence was very uncertain for all other interventions compared to placebo or no active treatment (intralesional dexamethasone injections, pentoxifylline, hydrocortisone plus hyaluronidase, physiotherapy). Burning sensation Antioxidants probably reduce burning sensation visual analogue scale (VAS) scores at less than three months (MD -30.92 mm, 95% CI -31.57 to -30.27; 1 study, 400 participants; moderate-certainty evidence), at three to six months (MD -70.82 mm, 95% CI -94.39 to -47.25; 2 studies, 500 participants; moderate-certainty evidence) and at more than six months (MD -27.60 mm, 95% CI -36.21 to -18.99; 1 study, 90 participants; moderate-certainty evidence). The evidence was very uncertain for the other interventions that were compared to placebo and measured burning sensation (intralesional dexamethasone, vasodilators). Adverse effects Fifteen studies reported adverse effects as an outcome. Six of these studies found no adverse effects. One study evaluating abdominal dermal fat graft reported serious adverse effects resulting in prolonged hospital stay for 3/30 participants. There were mild and transient general adverse effects to systemic drugs, such as dyspepsia, abdominal pain and bloating, gastritis and nausea, in studies evaluating vasodilators and antioxidants in particular. AUTHORS' CONCLUSIONS: We found moderate-certainty evidence that antioxidants administered systemically probably improve mouth opening slightly at three to six months and improve burning sensation VAS scores up to and beyond six months. We found only low/very low-certainty evidence for all other comparisons and outcomes. There was insufficient evidence to make an informed judgement about potential adverse effects associated with any of these treatments. There was insufficient evidence to support or refute the effectiveness of the other interventions tested. High-quality, adequately powered intervention trials with a low risk of bias that compare biologically plausible treatments for OSF are needed. It is important that relevant participant-reported outcomes are evaluated.

Evaluation of possible role of fluoride in pathogenesis of oral submucous fibrosis: A pilot study.

BACKGROUND: Oral submucous fibrosis (OSMF) is a potentially malignant disorder. Although areca nut chewing is an established risk factor, its low prevalence among nut chewers indicates additional factors likely facilitates pathogenesis. We recently demonstrated high fluoride levels in smokeless tobacco products and hypothesized a potential pathological role of fluoride in OSMF. Further exploring this novel role, this study compared fluoride levels in tissue, serum, and saliva samples from OSMF patients and healthy controls. METHODS: The ethically approved study included 25 clinically confirmed OSMF patients and 25 healthy matched controls. OSMF cases underwent buccal mucosal incisional biopsy, while controls had buccal mucosa tissue sampling during third molar removal. Fasting venous blood and unstimulated saliva were collected. Fluoride levels were analysed using ion chromatography and expressed as median (IQR). RESULTS: OSMF cases showed significantly higher fluoride concentrations compared with controls in tissue biopsies (30.1 vs. 0 mg/kg, p < 0.0001), serum (0.4 vs. 0 mg/L, p = 0.005) and saliva (1.3 vs. 0 mg/L, p < 0.0001). Majority (68%) of controls had undetectable fluoride levels across all samples. Tissue fluoride weakly correlated with OSMF severity (r = -0.158, p = 0.334). CONCLUSION: The preliminary findings demonstrated increased tissue fluoride levels in OSMF patients compared with healthy controls. Along with a previous study showing high fluoride content in smokeless tobacco products, these findings provided early evidence suggesting fluoride could play a contributory role in OSMF pathogenesis. Further large-scale investigation is warranted to definitively establish whether the association between fluoride exposure and OSMF is indicative of causation.

Antifibrotic effect of silymarin on arecoline-induced fibrosis in primary human buccal fibroblasts: an in silico and in vitro analysis.

BACKGROUND: This study aimed to assess silymarin's anticancer and antifibrotic potential through in silico analysis and investigate its impact on in vitro arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS & RESULTS: The study utilized iGEMDOCK for molecular docking, evaluating nine bioflavonoids, and identified silymarin and baicalein as the top two compounds with the highest target affinity, followed by subsequent validation through a 100ns Molecular Dynamic Simulation demonstrating silymarin's stable behavior with Transforming Growth Factor Beta. HBF cell lines were developed from tissue samples obtained from patients undergoing third molar extraction. Arecoline, a known etiological factor in oral submucous fibrosis (OSMF), was employed to induce fibrogenesis in these HBFs. The inhibitory concentration (IC50) of arecoline was determined using the MTT assay, revealing dose-dependent cytotoxicity of HBFs to arecoline, with notable cytotoxicity observed at concentrations exceeding 50µM. Subsequently, the cytotoxicity of silymarin was assessed at 24 and 72 h, spanning concentrations from 5µM to 200µM, and an IC50 value of 143µM was determined. Real-time polymerase chain reaction (qPCR) was used to analyze the significant downregulation of key markers including collagen, epithelial-mesenchymal transition (EMT), stem cell, hypoxia, angiogenesis and stress markers in silymarin-treated arecoline-induced primary buccal fibroblast cells. CONCLUSION: Silymarin effectively inhibited fibroblast proliferation and downregulated genes associated with cancer progression and EMT pathway, both of which are implicated in malignant transformation. To our knowledge, this study represents the first exploration of silymarin's potential as a novel therapeutic agent in an in vitro model of OSMF.

Stromal thrombospondin 1 suppresses angiogenesis in oral submucous fibrosis.

A decline in mucosal vascularity is a histological hallmark of oral submucous fibrosis (OSF), a premalignant disease that is largely induced by betel quid chewing. However, the lack of available models has challenged studies of angiogenesis in OSF. Here, we found that the expression of thrombospondin 1 (THBS1), an endogenous angiostatic protein, was elevated in the stroma of tissues with OSF. Using a fibroblast-attached organoid (FAO) model, the overexpression of THBS1 in OSF was stably recapitulated in vitro. In the FAO model, treatment with arecoline, a major pathogenic component in areca nuts, enhanced the secretion of transforming growth factor (TGF)-ß1 by epithelial cells, which then promoted the expression of THBS1 in fibroblasts. Furthermore, human umbilical vein endothelial cells (HUVECs) were incorporated into the FAO to mimic the vascularized component. Overexpression of THBS1 in fibroblasts drastically suppressed the sprouting ability of endothelial cells in vascularized FAOs (vFAOs). Consistently, treatment with arecoline reduced the expression of CD31 in vFAOs, and this effect was attenuated when the endothelial cells were preincubated with neutralizing antibody of CD36, a receptor of THBS1. Finally, in an arecoline-induced rat OSF model, THBS1 inhibition alleviated collagen deposition and the decline in vascularity in vivo. Overall, we exploited an assembled organoid model to study OSF pathogenesis and provide a rationale for targeting THBS1.