Evaluation of Presence of Enterococcus faecalis in Root Canals of Deciduous Molars with Necrotic Pulp by Agar Culture and Polymerase Chain Reaction.

Objective: To assess the presence of Enterococcus faecalis in root canals of deciduous molars with necrotic pulp by agar culture and polymerase chain reaction (PCR) assay. Materials and methods: This is an experimental study, where a total of 120 endodontic samples were taken from deciduous molars with necrotic pulps. The presence of Enterococcus faecalis was assessed by culture, using Enterococcus confirmatory agar, and by PCR assay. Statistical analysis of the results was performed using McNemar's test. Results: The presence of Enterococcus faecalis was detected in 20 samples (16.67% of total) by microbial culture and in 45 samples (37.5% of total) by PCR assay, with a statistically significant difference between the two methods (p < 0.001). Microbial culture and PCR both detect Enterococcus faecalis, with the latter detecting an additional 25 positive samples. Conclusion: In this study, PCR assay was significantly more sensitive than agar culture method in detecting the presence of Enterococcus faecalis in root canals of deciduous molars with necrotic pulp, that is, 37.5% of all samples. Clinical significance: Importance of presence of Enterococcus faecalis in necrotic pulps of deciduous teeth, as it is primarily responsible for failure of endodontic treatment, thus helping clinicians to advocate the use of local drug delivery in primary teeth endodontics and also aids clinicians in choosing the most effective intracanal medication. How to cite this article: Nalawade TM, Bhat KG, Kale AD, et al. Evaluation of Presence of Enterococcus faecalis in Root Canals of Deciduous Molars with Necrotic Pulp by Agar Culture and Polymerase Chain Reaction. Int J Clin Pediatr Dent 2023;16(6):816-819.

Role of cancer-associated fibroblasts in oral squamous cell carcinomas, surgical margins, and verrucous carcinomas: An immunohistochemical study.

Background and Aim: Cancer-associated fibroblasts (CAFs) are among the key tumor microenvironment components that determine tumor invasion, progression, and resistance to cancer therapeutics. Histologically normal mucosa adjacent to oral squamous cell carcinoma (OSCC) has been shown to harbor CAFs which aid in the loco-regional recurrence of the lesion. Verrucous carcinoma (VC), a low-grade variant of squamous cell carcinoma, has a better clinical outcome. However, few VCs show an aggressive biological course and necessitate wide excision with strict follow-up. Scarce literature is available regarding the role of CAFs in VCs. Thus, our study aimed to evaluate the frequency of CAFs in OSCC, normal mucosa adjacent to OSCC, and VC. Methods: Thirty cases of squamous cell carcinoma, normal mucosa adjacent to OSCC, and VC each were included in the study. The sections were stained with an antibody against alpha-smooth muscle actin protein and CAF frequency was evaluated. Results: The CAF frequency was highest in squamous cell carcinoma, followed by VC, and least in normal mucosa adjacent to OSCC (P<0.001). Conclusion: CAF frequency progressively increases with an increase in the grade or biological behavior of the lesion. Thus, screening CAF frequency in these benign and malignant oral lesions is necessary for better treatment outcomes. Relevance for Patients: The immunohistochemical screening for CAFs in OSCC and VC can serve as an integrated approach for the development of a directed treatment plan that leads to a better patient prognosis. Routine assessment of CAF frequency in surgical margins can serve as an adjunct in determining clear margins and possible locoregional recurrence. Furthermore, target therapy for CAFs can be used to minimize possible recurrence and distant metastasis.

Role of programmed cell death 4 in myofibroblast differentiation in oral submucous fibrosis.

Background: Fibrosis is an uncontrolled healing process, led by persistent differentiation of fibroblast to alpha-smooth muscle actin (αSMA) positive activated fibroblast or myofibroblast. Oral submucous fibrosis (OSMF) is one such condition that is associated with areca nut use. Recently, Programmed Cell Death 4 (PDCD4), a pro-apoptotic marker, has been shown to modulate fibroblast differentiation in various organ fibrosis. The present study aimed to evaluate the role of PDCD4 in the regulation of fibroblast differentiation in OSMF. Materials and Methods: Paraffin-embedded tissue sections from 45 cases of the normal oral mucosa, early OSMF and advanced OSMF were examined for PDCD4 and αSMA expression by immunostaining. Co-expression of PDCD4 and αSMA in fibroblasts was examined using Spearman's correlation test. Results: The stromal fibroblasts showed minimal expression of αSMA in the normal mucosa and early OSMF, while advanced OSMF groups demonstrated a higher frequency of αSMA myofibroblasts. The PDCD4 expression was noted in the normal stromal fibroblasts. However, this expression appeared to progressively reduce with an increasing grade of OSMF. Thus, a negative correlation was noted between stromal PDCD4 and αSMA expression with progressive OSMF. Conclusion: This study demonstrated a putative role for PDCD4 in oral fibrosis consistent with its role in other tissues. The lack of PDCD4 expression with increasing myofibroblast expression in OSMF suggests that targeting its dysregulation may be an attractive translational therapeutic target.

Isolation, culture and characterization of primary cell lines of human buccal mucosal fibroblasts: A combination of explant enzamytic technique.

BACKGROUND: The cell culture technique has become a routine and a popular method for its wide applications in the field of cell biology and biotechnology and in medical research. Isolation of primary cells over the cancer cells is an essential component of cell culture technology as they are the reliable source to understand normal physiological, morphological and molecular process of human cells. As fibroblasts are the prominent cells of the connective tissue of oral mucosa, many disease entities and histogenesis are linked to fibroblasts. Culture of oral fibroblast cells helps the oral biologists and researchers to study the morphological and molecular process in the oral diseases. AIM: The aim of our experiment is to isolate and culture the human buccal mucosal fibroblast cells from healthy individuals using a combination of explant-enzymatic method and characterization of the cells by short tandem repeat (STR) profiling. MATERIALS AND METHODS: The tissue samples were collected from healthy individuals undergoing routine impacted third molar extraction. A combination of explant-enzymatic technique was used for the isolation from the tissue samples. The cells were further subcultured, maintained and stored as per the standard protocols. Thus, to confirm the oral fibroblasts of human origin and its uniqueness, they were characterized using STR profiling. RESULTS AND CONCLUSION: Using the combination technique, we were successful in isolating the cells at a faster rate by detachment of cells on day 3 and confluency on day 10. The morphological assessment and STR profiling further confirmed that the isolated cell lines resemble human fibroblast cells.

Roles of the matricellular protein Tenascin-C in T-lymphocyte trafficking and etiopathogenesis of Oral Lichen Planus.

OBJECTIVE: This study was aimed at examining the role of Tenascin-C in T cell trafficking in Oral Lichen Planus (OLP). DESIGN: For the in vivo immunohistochemical analyses, 115 OLP samples were collected from patients and immunostaining was performed. The intensity and distribution of TN-C expression were quantified and correlated with histological analyses of basement membrane integrity and presence of inflammatory infiltrate. For the in vitro study, TN-C and collagen were coated on culture plates and migration of T lymphocytes was assessed. RESULTS: TN-C immunoexpression was increased in terms of both distribution and intensity along the basement membrane zone. These changes were significantly associated with basement membrane duplication (distribution p < 0.002 and intensity p < 0.001) and bands of inflammation (distribution p < 0.002 and intensity p < 0.001) assessed by Chi-square test. T lymphocytes demonstrated significant migration towards TN-C as compared to collagen (n = 3, p < 0.05). CONCLUSIONS: These findings indicate TN-C may have a key role in promoting T cell migration at the epithelial-mesenchymal junction in OLP. These observations suggest TN-C could be a good target for therapeutic intervention, either in itself or synergistically with anti-inflammatory directed strategies in this chronic disease management.

Role of immunoreactive patterns of lymph nodes in neck dissection cases of oral squamous cell carcinoma: a clinical and histopathological study.

OBJECTIVES: Metastasis in oral squamous cell carcinoma (OSCC) can occur in a variety of ways, and draining lymphatics and lymph nodes serve as a common route. Prior to metastasis, lymph nodes elicit an immune response to either wall off or create a favorable environment for homing of tumor cells. This immune response to tumor stimuli is visualized by recognizing various immunoreactive patterns exhibited by the lymph node. The present study aims to evaluate the role of immuno-morphologic patterns of the lymph node in neck dissection for cases of OSCC. MATERIALS AND METHODS: Our retrospective study included 50 neck dissection cases of OSCC and a total of 1,078 lymph nodes. The grades of primary tumors with eight different immunoreactive patterns were compared. Vascularity and metastasis in lymph nodes were also evaluated. RESULTS: The lymphocyte predominant pattern was the most common immunoreactive pattern found in 396 of 1,078 lymph nodes. Patterns of lymphocyte predominant (P=0.0005), sinus histiocytosis (P=0.0500), paracortical hyperplasia (P=0.0001), cortical hyperplasia (P=0.0001), and increased vascularity (P=0.0190) were significantly associated with tumor grade. CONCLUSION: The present study adds to the understanding of lymph node immunoreactivity patterns and their correlation with tumor grade. We recommend further study of lymph node patterns for all sentinel lymph node biopsies and routine neck dissections for OSCCs.

Salivary calcium as a diagnostic tool for screening of osteoporosis in postmenopausal women.

BACKGROUND: Women's health undergoes physiological, pathological and psychological changes after menopause. Reduced estrogen levels have been implicated in the pathogenesis of osteoporosis in postmenopausal women. Estrogen is also known to affect the salivary gland functions. To understand the association between serum estrogen, osteoporosis and salivary calcium, the present cross-sectional study was undertaken. AIM: The aim of this study is to determine salivary calcium levels and its use for the diagnosis of osteoporosis in postmenopausal women. METHODOLOGY: Ninety individuals divided into three groups of healthy controls, pregnant women and postmenopausal women were selected. Serum estrogen, salivary calcium and bone mineral density (BMD) at the heel region were estimated. Statistical analysis using the Mann-Whitney U-test was done to compare the results within the groups. RESULTS: Mean estrogen levels were 115.8 ± 80.18 pg/mmol in control group, 7729.4 ± 907.6 pg/mmol in pregnant group and 51.2 ± 74.51 pg/mmol in postmenopausal group, respectively. The mean salivary calcium in control, pregnant and postmenopausal groups was 3.12 ± 0.63, 3.19 ± 0.62 and 7.12 ± 0.79 µg/dl, respectively. Paired comparison within the groups showed high statistical significance (P = 0.0000) in the salivary calcium levels. The mean BMD of -2.3 (standard deviation [SD] ± 0.83) in the postmenopausal group was significantly lower than -0.6 (SD ± 0.99) and -0.2 (SD ± 1.42) of pregnant and control groups, respectively. CONCLUSION: A negative correlation was found between estrogen and bone density. A significant difference in salivary calcium was noted in the study groups, highlighting the role of salivary calcium in the detection of early bone changes in postmenopausal women.

Stature estimation from regression analysis of facial anthropometry in Indian population.

BACKGROUND: Stature or body height is one of the most important and useful anthropometric parameters which determines the physical identity of an individual. As craniofacial structures have the advantage of being composed largely of hard tissue which is relatively indestructible, the careful study of these can enable reliable determination of stature of the person in life. Studies pertaining to stature estimation from facial measurements are limited in an Indian population. The present investigation attempts to estimate stature from anthropometric dimensions of face. MATERIALS AND METHODS: The material for the present study comprises 361 Indian students (151 males and 210 females) in the age range of 21-45 years. Stature and six facial measurements were taken on each participant following standard methods and techniques. Karl Pearson's correlation coefficient and linear regression were done to estimate stature. RESULTS: The results indicate that facial measurements are strongly and positively correlated (P < 0.001) with stature. The accuracy of the computed equations was further tested on 50 randomly selected study participants of each group, which shows close approximation of actual and estimated stature. CONCLUSION: Within the limits of this study, we conclude that facial dimensions can be used as a supplementary approach for the estimation of stature but with caution, as these are population-specific approach.

Assessment of morphological parameters associated with neural invasion in oral squamous cell carcinoma.

AIMS: Neural invasion (NI) is a form of tumor spread distinct from lymphatic and vascular invasion. It has been correlated with aggressive behavior, disease recurrence and increased morbidity and mortality. Despite the importance of NI as a prognostic indicator, the mechanism and associated factors are poorly understood. Hence, the aim of this study was to determine morphological parameters associated with NI in oral squamous cell carcinoma (OSCC). METHODOLOGY: One hundred and five archival specimens of patients with primary OSCC who underwent surgical excision and radical neck dissection were included in the study. The presence of NI was analyzed in slides and correlated with clinical as well as morphological parameters using univariate and multivariate analysis. RESULTS: NI was identified in 31 cases (29%) of OSCC. NI was significantly associated with tobacco habit, lymphovascular invasion and positive surgical margins. Multivariate analysis further emphasized these factors to be significant risk factors for NI. Peri-NI and intra-NI significantly associated with the size of the tumor, while the distance of invasion was associated with advanced stage. CONCLUSION: NI is associated with multiple morphological parameters and its identification may have a significant impact on the management and prognostic evaluation of OSCC.

Tumor budding is a potential histopathological marker in the prognosis of oral squamous cell carcinoma: Current status and future prospects.

Tumor budding (TB) is a histopathologically evident feature that represents a scattered pattern of invasion consisting of isolated single tumor epithelial cells or tumor cells in small clusters (up to 5 cells) seen primarily at the invasive front dispersed within the stroma for variable distance. Presence of TB has been linked with lymph node metastasis, recurrence, distant metastasis and reduced survival in numerous cancers including oral squamous cell carcinoma (OSCC). Tumor buds are also considered as histopathological markers of epithelial-mesenchymal transition which is a molecular process implicated as a hallmark for invasion and metastasis. This review gives an overview of the current evidence regarding TB assessment in OSCC and its future prospects.

Assessment of cell block technique in head and neck pathology diagnoses: A preliminary study.

OBJECTIVE: The head and neck region is a composite site made of multiple tissue components. These tissues when affected by disease or pathology present with an array of changes in the tissue architecture and pattern. It is essential to visualize the cellular details and tissue patterns for accurate diagnosis and treatment planning. Aspiration cytology primarily makes use of the cellular details for diagnosing lesions of the head and neck. Despite the promising results, its use is still limited in certain cases of the head and neck. The reason implicated could be the indiscernible appearance of cells in the absence of tissue integrity. In this regard, cell blocks are known to facilitate the visualization of the cytomorphological as well as the tissue arrangement patterns. Thus, the present study was designed to evaluate the role of cell block cytology in the diagnosis of various lesions of the head and neck. METHODS: Odontogenic lesions, epithelial carcinomas and connective tissue pathology of the head and neck origin were included in the study (n = 45). Aspiration cytology smears and cell block diagnosis were compared with tissue biopsy diagnosis for determining their sensitivity (%) and diagnostic efficacy. RESULTS: Cell blocks showed distinct preservation of the architectural pattern. In case of fluid-filled lesions, the contents were preserved and correlated with the tissue biopsy results. The results of cell blocks were similar to that of tissue biopsy in majority of the cases (95.56%). CONCLUSION: We recommend using cell blocks as a part of routine laboratory practice for all head-neck cases.

Modified Alcohol-Formalin Cell Block Technique in Head and Neck Pathology Diagnosis.

OBJECTIVES: Cell block preparation is a recognized technique in histopathological diagnosis. This technique aids in maintaining an intact architecture and reducing the diagnostic errors associated with fine-needle aspiration cytology. Numerous techniques have been put forth, but the need for an optimal technique for routine use in the laboratory still persists. The aim of our study was to establish a cell block technique which aids in the accurate diagnosis of head and neck pathology. METHODOLOGY: A modified cell block technique was developed using alcohol-formalin as a fixative. Forty fine-needle aspiration fluids from clinically and radiologically diagnosed cases of head and neck pathology were used as samples. The cell block sections were compared with the cytology smears to determine the utility of the technique. RESULTS: The cell blocks presented with better preservation of the architectural framework and enabled a quick diagnosis. Cellular clumping was negligible, and nuclear as well as cellular details were maintained similar to tissue sections. It led to the integration of conventional techniques using 10% neutral buffered formalin with that of the 10% alcohol-formalin technique. CONCLUSION: Modified cell block technique can be used as a simple and effective tool in the routine diagnosis of head and neck pathology.

Validation of immunoexpression of tenascin-C in oral precancerous and cancerous tissues using ImageJ analysis with novel immunohistochemistry profiler plugin: An immunohistochemical quantitative analysis.

BACKGROUND: Immunohistochemistry (IHC) is a molecular technique that has grown tremendously over the years. However, the assessment is only qualitative which is subjective and causes errors. Due to this limitation, several excellent markers have not gained importance and reached clinical trials. Hence, we aimed to quantify IHC by ImageJ analysis with a novel IHC profiler plugin. ImageJ has not been tried in oral precancerous tissues with minimal attempt for matrix markers. AIM: This study aimed to validate the quantification of immunoexpression of tenascin-C (TN-C) in oral precancerous tissues and oral squamous cell carcinoma (OSCC) using ImageJ software with IHC profiler plugin. MATERIALS AND METHODS: After IHC staining for TN-C and image acquisition, ImageJ analysis was performed as per the standard recommended algorithm. Assessment was done by two observers by blinding the histopathological diagnosis. The immunoscore was assessed for interobserver variability using Kohen's kappa statistics. RESULTS: All our cases were in agreement and found to be statistically significant with P < 0.005. Moderate agreement was for mild dysplasia, moderate dysplasia and oral lichen planus. Substantial agreement was for oral submucous fibrosis and OSCC and almost perfect agreement noted for cases of severe dysplasia. CONCLUSION: IHC can now be quantified using freely downloadable software ImageJ analysis in oral precancerous tissues and OSCC. This software with good threshold control can quantify matrix marker such as TN-C. Hence, herewith, we propose that IHC markers should be quantified using ImageJ by our entire oral pathology fraternity so as to have a standard immunoscore for all markers.

Oral Carcinoma Cuniculatum: A New Entity in the Clinicopathological Spectrum of Oral Squamous Cell Carcinoma.

Carcinoma cuniculatum is principally recognized as a variant of carcinoma involving foot. The World Health Organization (WHO) recognizes Oral Carcinoma Cuniculatum (OCC) as a distinct and rare clinicopathological variant of Oral Squamous Cell Carcinoma (OSCC). OCC is confused clinically and histologically with Verrucous Carcinoma (VC) and is often misdiagnosed as either VC or OSCC. To best of our knowledge, till date, only 50 cases of this tumour have been reported in oral cavity (including the present case) and only limited number of cases have been reported from Indian subcontinent. Pathognomonic feature of OCC is proliferation of stratified squamous epithelium and its infiltration into underlying stroma forming a complex pattern of keratin cores and keratin filled crypts. These complex crypts give it a likeness of rabbit burrow hence, the name cuniculatum (cuniculatus='rabbit warren'). The report aims to present a case of OCC of mandibular gingiva, discuss its diagnostic features and highlight its differences from VC and OSCC.