A Novel Preclinical In Vitro 3D Model of Oral Carcinogenesis for Biomarker Discovery and Drug Testing

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents

Three-Dimensional Cell Culture Models to Investigate Oral Carcinogenesis: A Scoping Review

Three-dimensional (3-D) cell culture models, such as spheroids, organoids, and organotypic cultures, are more physiologically representative of the human tumor microenvironment (TME) than traditional two-dimensional (2-D) cell culture models. They have been used as in vitro models to investigate various aspects of oral cancer but, to date, have not be widely used in investigations of the process of oral carcinogenesis. The aim of this scoping review was to evaluate the use of 3-D cell cultures in oral squamous cell carcinoma (OSCC) research, with a particular emphasis on oral carcinogenesis studies. Databases (PubMed, Scopus, and Web of Science) were systematically searched to identify research applying 3-D cell culture techniques to cells from normal, dysplastic, and malignant oral mucosae. A total of 119 studies were included for qualitative analysis including 53 studies utilizing spheroids, 62 utilizing organotypic cultures, and 4 using organoids. We found that 3-D oral carcinogenesis studies had been limited to just two organotypic culture models and that to date, spheroids and organoids had not been utilized for this purpose. Spheroid culture was most frequently used as a tumorosphere forming assay and the organoids cultured from human OSCCs most often used in drug sensitivity testing. These results indicate that there are significant opportunities to utilize 3-D cell culture to explore the development of oral cancer, particularly as the physiological relevance of these models continues to improve

Gene expression profiling and molecular signaling of dental pulp cells in response to tricalcium silicate cements: a systematic review

Introduction: Signaling molecules and responding dental pulp stem cells are the 2 main control keys of dentin regeneration/dentinogenesis. The aim of this study was to present a systematic review investigating the gene expression of various dental pulp cells in response to different variants of tricalcium silicate cements. Methods: A systematic search of the literature was performed by 2 independent reviewers followed by article selection and data extraction. Studies analyzing all sorts of dental pulp cells (DPCs) and any variant of tricalcium silicate cement either as the experimental or as the control group were included. Results: A total of 39 articles were included in the review. Among the included studies, Pro Root MTA (Dentsply, Tulsa Dental, OK) was the most commonly used tricalcium silicate cement variant. The extracellular signal regulated kinase/mitogen-activated protein kinase pathway was the most commonly activated pathway to be identified, and similarly, dentin sialophosphoprotein osteocalcin dentin matrix acidic phosphoprotein 1, alkaline phos. phatase, bone sialoprotein, osteopontin, type I collagen, and Runx2 were the most commonly expressed genes in that order of frequency. Conclusions: Biodentine (Septodont Ltd, Saint Maur des Fausses, France), Bioaggregate (Innovative Bioceramix, Vancouver, BC, Canada), and mineral trioxide aggregate stimulate the osteogenic/odontogenic capacity of DPCs by proliferation, angiogenesis, and biomineralization through the activation of the extracellular signal regulated kinase 1/2, nuclear factor E2 related factor 2, p38, c-Jun N-terminal kinase mitogen-activated protein kinase, p42/p44 mitogen-activated protein kinase, nuclear factor kappa B, and fibroblast growth factor receptor pathways. When DPCs are placed into direct contact with tricalcium silicate cements, they show higher levels of gene activation, which in turn could translate into more effective pulpal repair and faster and more predictable formation of reparative dentin

Gene expression profiling and molecular signaling of various cells in response to tricalcium silicate cements: a systematic review

Introduction: The aim of this study was to present a systematic review investigating the gene expression of various cells (other than dental pulp cells) in response to different variants of tricalcium silicate cements (TSCs). Material and Methods: A systematic search of the literature was performed by two independent reviewers followed by article selection and data extraction. Studies analyzing any cell type except dental pulp stem cells and any variant of tri-calcium silicate cement either as the experimental or as the control group were included. Results: A total of 41 relevant articles were included in this review. Among the included studies, ProRoot MTA was the most commonly studied (69.1%) TSC variant, and 11 cell types were identified, with a majority of 13 articles investigating gene expression in osteoblasts. A total of 39 different genes/molecules expressed were found in the selected studies. The experimental group (irrespective of the TSC variant) was identified to express significantly increased gene expression than the control group (untreated) in all included studies. Discussion: Recent studies have provided useful insight into the gene expression and molecular signaling of various cells in response to TSCs, and new elements have been supplied on the pathways activated in this process. Conclusion: TSCs are capable of eliciting a favorable cellular response in periapical regeneration

A review on role of essential trace elements in health and disease

Elements are present in different forms in the nature, and these elements are very essential for the body to perform different functions. Trace elements are very important for cell functions at biological, chemical and molecular levels. These elements mediate vital biochemical reactions by acting as cofactors for many enzymes, as well as act as centers for stabilizing structures of enzymes and proteins. Some of the trace elements control important biological processes by binding to molecules on the receptor site of cell membrane or by alternating the structure of membrane to prevent entry of specific molecules into the cell. The functions of trace elements have a dual role. In normal levels, they are important for stabilization of the cellular structures, but in deficiency states may stimulate alternate pathways and cause diseases. These trace elements have clinical significance and these can be estimated using different analytical method

Molecular pathogenesis and diagnostic imaging of metastatic jaw tumors

Metastasis is the spread of malignant cells from a primary tumor to distant sites through lymphatics or blood vessels. Malignant lesions metastasizing to the oral and perioral region are a rarity indeed. Malignant lesions could metastasize to both soft tissue of oral cavity and the hard tissues of the jaws and recent meta-analysis showed that metastasis is more common in the jaws than oral soft tissues because of rich vascular supply. The incidence is very low when compared to the incidence of primary oral cancers; nevertheless, one has to include in the diagnostic workup, metastatic malignant lesions, when an irregular ill-defined radiolucency or radiodensity with ragged edges in noted. It could be a challenging task for a diagnostician, in cases with the presence and location of the primary tumor is unknown. Advanced oral imaging technologies and biochemical markers play a vital role in diagnosing such lesions

Histomorphometric analysis of vascularity in oral epithelial dysplasia

Context: Potentially malignant disorders such as leukoplakia and erythroplakia are often associated with dysplastic changes that have an increased risk for malignant transformation. Vascularity could have a role in the transformation of potentially malignant disorders to oral squamous cell carcinoma. Aims: The present study was aimed to assess the mean vessel area (MVA) of the blood capillaries in different grades of epithelial dysplasia by histomorphometric analysis, which could aid in predicting the malignant transforming potential as well as prognosis of potentially malignant disorders. Materials and Methods: The study consisted of 30 histologically proven epithelial dysplasia cases categorized into three groups of mild, moderate, and severe epithelial dysplasia based on the World Health Organization (WHO) 2005 criteria. Paraffin-embedded tissue sections of 3-4 microns were stained with Masson′s trichrome. Morphometric analysis of MVA was done using image analysis software 6.0. Statistical Analysis Used: Statistical analysis was done using the software Statistical Package for the Social Sciences (SPSS) 20.0. The mean and standard deviation of the three groups were analyzed using Kruskal-Wallis analysis of variance (ANOVA) test. Pairwise comparison was done using Mann-Whitney U test. P value <0.05 was considered to be statistically significant. Results: The MVA was highest in severe epithelial dysplasia (7575.16) followed by moderate (4070.4) and mild (4014.29) epithelial dysplasias. A high statistical significance was observed on comparison of the three groups (P 0.0004). On pairwise comparison, a statistical significance was observed between mild and severe epithelial dysplasias (P 0.0032) and moderate and severe epithelial dysplasias (P 0.0002). Conclusions: The MVA was increased as the grade of dysplasia progressed. This suggests that altered vascularity could play a role in the progression and malignant potential of dysplastic lesions and also in assessing the prognosis