Identification of Molecular Mechanisms of Ameloblastoma and Drug Repositioning by Integration of Bioinformatics Analysis and Molecular Docking Simulation.

Background: Ameloblastoma (AM) is a benign tumor locally originated from odontogenic epithelium that is commonly found in the jaw. This tumor makes aggressive invasions and has a high recurrence rate. This study aimed to investigate the differentially expressed genes (DEGs), biological function alterations, disease targets, and existing drugs for AM using bioinformatics analysis. Methods: The data set of AM was retrieved from the GEO database (GSE132474) and identified the DEGs using bioinformatics analysis. The biological alteration analysis was applied to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Protein-protein interaction (PPI) network analysis and hub gene identification were screened through NetworkAnalyst. The transcription factor-protein network was constructed via OmicsNet. We also identified candidate compounds from L1000CDS2 database. The target of AM and candidate compounds were verified using docking simulation. Results: Totally, 611 DEGs were identified. The biological function enrichment analysis revealed glycosaminoglycan and GABA (γ-aminobutyric acid) signaling were most significantly up-regulated and down-regulated in AM, respectively. Subsequently, hub genes and transcription factors were screened via the network and showed FOS protein was found in both networks. Furthermore, we evaluated FOS protein to be a therapeutic target in AMs. Candidate compounds were screened and verified using docking simulation. Tanespimycin showed the greatest affinity binding value to bind FOS protein. Conclusions: This study presented the underlying molecular mechanisms of disease pathogenesis, biological alteration, and important pathways of AMs and provided a candidate compound, Tanespimycin, targeting FOS protein for the treatment of AMs.

Exploring protein profiles and hub genes in ameloblastoma.

Ameloblastoma (AM) is a prominent benign odontogenic tumor characterized by aggressiveness, likely originating from tooth-generating tissue or the dental follicle (DF). However, proteomic distinctions between AM and DF remain unclear. In the present study, the aim was to identify the distinction between AM and DF in terms of their proteome and to determine the associated hub genes. Shotgun proteomics was used to compare the proteomes of seven fresh-frozen AM tissues and five DF tissues. Differentially expressed proteins (DEPs) were quantified and subsequently analyzed through Gene Ontology-based functional analysis, protein-protein interaction (PPI) analysis and hub gene identification. Among 7,550 DEPs, 520 and 216 were exclusive to AM and DF, respectively. Significant biological pathways included histone H2A monoubiquitination and actin filament-based movement in AM, as well as pro-B cell differentiation in DF. According to PPI analysis, the top-ranked upregulated hub genes were ubiquitin C (UBC), breast cancer gene 1 (BRCA1), lymphocyte cell-specific protein-tyrosine kinase (LCK), Janus kinase 1 and ATR serine/threonine kinase, whereas the top-ranked downregulated hub genes were UBC, protein kinase, DNA-activated, catalytic subunit (PRKDC), V-Myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein P53 and P21 (RAC1) activated kinase 1. When combining upregulated and downregulated genes, UBC exhibited the highest degree and betweenness values, followed by MYC, BRCA1, PRKDC, embryonic lethal, abnormal vision, Drosophila, homolog-like 1, myosin heavy chain 9, amyloid beta precursor protein, telomeric repeat binding factor 2, LCK and filamin A. In summary, these findings contributed to the knowledge on AM protein profiles, potentially aiding future research regarding AM etiopathogenesis and leading to AM prevention and treatment.

Downregulation of LAMB3 Altered the Carcinogenic Properties of Human Papillomavirus 16-Positive Cervical Cancer Cells.

Nearly all cervical cancer cases are caused by infection with high-risk human papillomavirus (HR-HPV) types. The mechanism of cervical cell transformation is related to the powerful action of viral oncoproteins and cellular gene alterations. Transcriptomic data from cervical cancer and normal cervical cells were utilized to identify upregulated genes and their associated pathways. The laminin subunit beta-3 (LAMB3) mRNAwas overexpressed in cervical cancer and was chosen for functional analysis. The LAMB3 was predominantly expressed in the extracellular region and the plasma membrane, which play a role in protein binding and cell adhesion molecule binding, leading to cell migration and tissue development. LAMB3 was found to be implicated in the pathway in cancer and the PI3K-AKT signaling pathway. LAMB3 knockdown decreased cell migration, invasion, anchorage-dependent and anchorage-independent cell growth and increased the number of apoptotic cells. These effects were linked to a decrease in protein levels involved in the PI3K-AKT signaling pathway and an increase in p53 protein. This study demonstrated that LAMB3 could promote cervical cancer cell migration, invasion and survival.

5-Methylcytosine immunohistochemistry for predicting cutaneous melanoma prognosis.

There is a correlation between DNA methylation and the diseased stage and poor survival. 5-methylcytosine (5-mC) is one of the epigenetic modifications of bases that researchers focus on. Staining with 5-mC immunohistochemistry was used to examine pathological samples taken from individuals diagnosed with cutaneous melanoma. Between Breslow levels 2 and 4, there was a significant difference in the H-score of 5-mC expression (p = 0.046). A significant reduction in 5-mC expression H-scores was seen in patients who were diagnosed with ulcers (p = 0.039). It was shown that patients with low 5-mC had a significantly worse overall survival rate (p = 0.027).

Distribution of Epstein-Barr virus in the oral cavity of Thais with various oral mucosal conditions.

Objectives: We aimed to examine the presence of EBV, EBV strains, and variants among 3 oral conditions including normal oral mucosa (NOM), oral potentially malignant disorders/oral cancer (OPMDs/OC) and non-OPMDs/OC in a group of Thais. Material and methods: Oral exfoliated cells were obtained from 315 participants living in the northeastern and central regions of Thailand. The participants were divided into 3 groups encompassing the NOM, the OPMDs/OC and the non-OPMDs/OC groups. The presence of EBV was first determined by PCR using primers for LMP1 gene. Subsequently, EBV strains of EBNA3c and variants based on LMP1 sequences were determined by real-time PCR. Results: The prevalence of EBV in OPMDs/OC, non-OPMDs/OC and NOM were 72.0 %, 56.2 %, and 27.2 % respectively. EBV type A, B and AB were found in 52.1 %, 32.1 % and 15.8 % of all positive samples, respectively. The percentage of participants with EBV type A was more prominent in the NOM group (72.0 %) compared to the non-OPMDs/OC (54.8 %) and the OPMDs/OC group (41.8 %) whereas EBV type B was higher in the OPMDs/OC group (35.8 %) compared to the non-OPMDs/OC (31.5 %) and the NOM (24.0 %) groups. Regarding EBV variants, 30-bp deletion LMP1 variant (del-LMP1) which is more associated with malignant transformation was predominately found in the OPMDs/OC (32.8 %) and the non-OPMDs/OC (38.4 %) groups compared to the NOM group (20.0 %). Conclusions: High frequency of EBV was demonstrated in the OPMDs/OC group. EBV type A was more predominant in the NOM group whereas EBV type B was more prevalent in the OPMDs/OC group. The del-LMP1 variant was more common in the OPMDs/OC and the non-OPMDs/OC groups.

CpG methylation changes in Alu repetitive sequences in normal aging due to diastolic hypertension in human dermal fibroblasts from the facial area.

Aging fibroblasts, an important factor contributing to skin aging, are affected by numerous mechanisms, including alterations in DNA methylation and age-related diseases. The current study aimed to investigate the role of Alu methylation in aging fibroblasts and hypertension. The Alu methylation levels in dermal fibroblasts obtained from patients of different ages and blood pressure status were analyzed using the combined bisulfite restriction analysis technique. An inverse correlation was observed between Alu methylation in dermal fibroblasts and patient age. Dermal fibroblasts from the high-normal diastolic blood pressure group had higher Alu methylation levels compared with those from the normal group. The findings of the present study suggest that Alu methylation alterations can be observed with chronological aging and hypertension, and are a potential aging marker or therapeutic target.

High 4-1BB Expression in PBMCs and Tumor Infiltrating Lymphocytes (TILs) in Patients with Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: 4-1BB is a costimulatory immune-activating molecule. Increased amounts of this protein have previously been found in the plasma of patients with oropharyngeal and oral cancer. Here, we focused on this molecule that functions as part of the immune system. We investigated 4-1BB in the peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) of patients with head and neck squamous cell cancer (HNSCC). MATERIALS AND METHODS: The expression level of 4-1BB in the PBMCs was determined using real-time polymerase chain reaction (PCR). The TIMER (Tumor Immune Estimation Resource) web server was utilized to approximate the 4-1BB level in HNSCC TILs. Moreover, 4-1BB immunohistochemistry (IHC) was used to validate TILs in four organs of HNSCC, including oral cancer (OC), oropharyngeal cancer (OPC), sinonasal cancer (SNC), and laryngeal cancer (LC), in both the tumor area and adjacent normal epithelium. The difference in 4-1BB expression levels in various groups was assessed using a Kruskal-Wallis test and an independent sample t-test. RESULTS: The level of 4-1BB expression in PBMCs was highest in OPC, followed by OC and healthy controls (HC). Significant differences were discovered between HC and OPC and between OC and OPC. Bioinformatics revealed a substantial correlation between 4-1BB expression level and lymphocyte infiltration in HNSCC, including B cells, CD8+ T cells, and CD4+ T cells. IHC validation in HNSCC tissue revealed that the average number of 4-1BB positive TILs in all four HNSCC subtypes was considerably greater than the number of lymphocytes seen in adjacent normal tissue. Interestingly, the number of lymphocytes that were 4-1BB positive increased in relation to the TIL level. CONCLUSION: A higher number of 4-1BB expression levels were found in the PBMCs and TILs of HNSCC patients, implying that 4-1BB may be a promising approach for HNSCC patients to improve their immune function. It is important to study and create a treatment that uses 4-1BB medicine as well as existing drugs.

Evaluation of P53 immunostaining in patients with cutaneous melanoma.

P53 is a tumor suppressor gene that is mutated in numerous types of cancer. The aim of the present study was to determine the frequency of this mutation in cutaneous melanomas and to conduct clinicopathological characteristics and clinical outcome association analyses with the P53 mutation. P53 immunohistochemical staining was used as a surrogate marker for P53 mutation analysis to assess P53 status. In the present study, 50 pathological samples of cutaneous melanoma from 2012 to 2018 at Chulalongkorn University (Bangkok, Thailand), were subjected to anti-P53 immunohistochemistry, followed by an examination of the association between P53 statuses and clinical and pathological characteristics, along with clinical outcomes. A positive staining for anti-P53 antibody was detected in 30% of patients (15/50) with cutaneous melanomas. Positivity was significantly associated with female sex, nodular histological subtype and Breslow level 4. Cox regression analysis revealed that an age >65.5 years and Breslow grade 4 disease were associated with mortality. The Kaplan-Meier curve revealed a shorter duration of recurrence time in the P53 mutation than P53 wild type. In the present study, P53 mutations in specific cases of cutaneous melanoma were identified. Notably, patients who were older and/or had a Breslow score of 4 exhibited an increased risk of mortality. These findings suggested the potential involvement of P53 mutations in cutaneous melanoma, highlighting the necessity for further investigations to improve understanding of their roles.

Protein expression analysis for predicting recurrent laryngeal squamous cell carcinoma.

The expression of a number of proteins plays a major role in predicting recurrent laryngeal squamous cell carcinoma (LSCC). Thus, the aim of the present study was to investigate the expression of 16 selected proteins as prognostic indicators for recurrent and non-recurrent LSCC. Samples from a total of 41 patients with LSCC were investigated by immunohistochemistry. Digital image analysis was performed, and various associated factors were calculated. Histoscore (H-score) and receiver operating characteristic curves were used to divide protein expression in high and low for predicting disease recurrence. Disease-free survival (DFS) curves, crude hazard ratios (HRs) and adjusted HRs were analyzed and compared. Significantly different H-scores were found between the recurrent and non-recurrent groups in terms of pRb and c-Met expression. pRb was expressed at high levels in recurrent LSCC, while c-Met was expressed at low levels. Patients with low pRb expression had a longer DFS than those with high pRb expression (log-rank χ2, 5.161; P=0.023). Patients with high c-Met expression had a longer DFS than those with low c-Met expression (log-rank χ2, 6.441; P=0.011). Moreover, patients with high pRb expression and low c-Met expression had the shortest DFS (log-rank χ2, 11.827; P=0.008). Differentiated histological factors had an impact on the risk of recurrence (Cox regression test; crude HR, 9.53; 95% confidence interval, 1.214-74.819; P=0.032). The present study demonstrated that the grading of differentiated squamous cell carcinoma, pRb and c-Met expression are the most useful prognostic factors for the prediction of recurrent LSCC. These might be further applied as potential markers for clinical use.

Diagnostic Accuracy of Immunohistochemistry for HER2-Positive Breast Cancer.

OBJECTIVE: Currently, human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells are diagnosed under the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2018 guidelines. The guideline combined the results of in situ hybridization (DISH) and immunohistochemistry (IHC) techniques. The IHC technique is easy, cheap, and suitable for developing country. Therefore, in this study, we validated the use of IHC alone compared to the results of HER2 amplification under ASCO/CAP 2018 guidelines in diagnosed HER2 positive breast cancer cells. METHODS: A total of 510 breast cancer tissue samples from Rajavithi Hospital in Bangkok, Thailand, from January 1st, 2022, to May 31st, 2023, were analyzed by IHC, followed by dual ISH (DISH). We selected 58 samples of IHC equivocal (score 2+) and 98 samples of IHC positive (score 3+) to analyze the diagnostic values by comparing them to the results of HER2 amplification. RESULTS: The HER2 IHC score was found to agree with HER2 amplification with a sensitivity of 87.96%, a specificity of 93.75%, a positive predictive value of 96.94%, a negative predictive value of 77.59%, a positive likelihood ratio of 14.07, a negative likelihood ratio of 0.13, and an accuracy of 89.74%. CONCLUSION: The promising outcomes suggest that a positive IHC test result (score 3+) could potentially stand alone for patients with breast cancer undergoing anti-HER2 treatment, even without DISH confirmation.

Efficiency of CIN2+ Detection by Thyrotropin-Releasing Hormone (TRH) Site-Specific Methylation.

Cervical cancer screening typically involves a Pap smear combined with high-risk human papillomavirus (hr-HPV) detection. Women with hr-HPV positivity but normal cytology, as well as those with precancerous abnormal cytology, such as low-grade squamous intraepithelial lesions (LSIL) and high-grade SIL (HSIL), are referred for colposcopy and histology examination to identify abnormal lesions, such as cervical intraepithelial neoplasia (CIN) and cervical cancer. However, in order to enhance the accuracy of detection, bioinformatics analysis of a microarray database was performed, which identified cg01009664, a methylation marker of the thyrotropin-releasing hormone (TRH). Consequently, a real-time PCR assay was developed to distinguish CIN2+ (CIN2, CIN3, and cervical cancer) from CIN2- (CIN1 and normal cervical epithelia). The real-time PCR assay utilized specific primers targeting methylated cg01009664 sites, whereas an unmethylated reaction was used to check the DNA quality. A cut-off value for the methylated reaction of Ct < 33 was established, resulting in improved precision in identifying CIN2+. In the first cohort group, the assay demonstrated a sensitivity of 93.7% and a specificity of 98.6%. In the cytology samples identified as atypical squamous cells of undetermined significance (ASC-US) and LSIL, the sensitivity and specificity for detecting CIN2+ were 95.0% and 98.9%, respectively. However, when self-collected samples from women with confirmed histology were tested, the sensitivity for CIN2+ detection dropped to 49.15%, while maintaining a specificity of 100%. Notably, the use of clinician-collected samples increased the sensitivity of TRH methylation testing. TRH methylation analysis can effectively identify women who require referral for colposcopy examinations, aiding in the detection of CIN2+.

Differential expression of PEA3 in odontogenic cysts and tumors.

BACKGROUND: PEA3 transcription factor has been identified as a downstream target of the MAPK and PI3K pathways, and PEA3 overexpression has been observed in a variety of tumor types. We aimed to evaluate PEA3 expression in odontogenic cysts and tumors and compare the expression among odontogenic lesions. In addition, the correlations between PEA3 expression and clinicopathological characteristics of conventional ameloblastoma and unicystic ameloblastoma were investigated. METHODS: This study was performed on 165 samples of odontogenic cysts and tumors including 20 dentigerous cysts, 20 odontogenic keratocysts, 16 adenomatoid odontogenic tumors, 5 ameloblastic fibromas, 45 unicystic ameloblastomas, and 59 conventional ameloblastomas. The sections were immunohistochemically stained with mouse monoclonal anti-PEA3 antibody and PEA3 expression was evaluated as the immunoreactive score. RESULTS: PEA3 expression was absent in all dentigerous cysts (DCs) and odontogenic keratocysts, while all adenomatoid odontogenic tumors showed either no (75%) or low (25%) expression of PEA3. Most of the ameloblastic fibromas (60%) displayed no PEA3 expression. A high expression of PEA3 was observed in a substantial number of unicystic ameloblastomas (48.9%) and conventional ameloblastomas (49.2%) in our study. PEA3 expression in DCs, odontogenic keratocysts and adenomatoid odontogenic tumors were significantly different from that in conventional ameloblastomas and that in unicystic ameloblastomas (p < 0.05). The expression of PEA3 was significantly different in the age groups of unicystic ameloblastomas and histological subtypes of conventional ameloblastomas (p < 0.05). CONCLUSION: PEA3 overexpression is predominant in unicystic ameloblastomas and conventional ameloblastomas compared to other odontogenic lesions, indicating a pivotal role of PEA3 as a downstream effector of MAPK pathway in these two odontogenic lesions.

Analysis of Epstein-Barr Virus Infection in Oral Potentially Malignant Disorders and Oral Cancer: A Cross-Sectional Study.

Aims and Objectives: The primary objective of this study is to determine the prevalence of Epstein-Barr virus (EBV) in oral potentially malignant disorders (OPMDs) and oral cancer (OC) in a group of Thais using polymerase chain reaction (PCR) and Epstein-Barr encoding regions (EBERs) in situ hybridization (ISH). The secondary objective is to investigate the risk factors of OC and the association between the presence of EBV and risk factors of OC/site of oral lesions. Materials and Methods: Sixty-one participants attending the screening project for OC and OPMDs at the Northeastern district hospitals of Thailand were recruited. Information related to risk factors and biopsy tissues for histopathological diagnosis was collected. Sixty-seven paraffin tissue blocks, including 52 OPMDs and 15 OC specimens, were investigated for EBV infection, using PCR analysis with latent membrane protein-1 (LMP-1) primer and EBERs ISH. Pearson's Chi-square or Fisher's exact test was used to analyze the differences in variables between participants with OPMDs and OC, as appropriate. The association between EBV infection and related risk factors was analyzed using logistic regression with a significant level at 0.05. Results: Using PCR analysis, 8 of 67 specimens (11.94%) were positive for LMP-1. Three cases of OPMDs were positive for both LMP-1 PCR and EBERs ISH. Regarding risk factors of OC, the two most common risk factors were betel nut chewing (52.46%) and working in sunlight (42.62%). The habit of taking alcohol was significantly different between the OC and the OPMDs groups (p = 0.009). The association between LMP-1 and the lesion at the tongue was statistically significant, with odds ratio = 4.900 (95% confidence interval = 1.046-22.943; p = 0.044). Conclusions: The prevalence of EBV infection in this group of participants was low. However, OPMDs at the tongue exhibited a significant association with EBV infection.

Identification of Potential Molecular Mechanisms and Prognostic Markers for Oral Squamous Cell Carcinoma: A Bioinformatics Analysis.

Aims and Objectives: The goal of this study was to uncover crucial biochemical pathways, prognostic indicators, and therapeutic targets in patients with oral cancer in order to enhance therapy strategies. Materials and Methods: Five gene expression omnibus datasets were analyzed by using bioinformatics approaches to identify differentially expressed genes (DEGs). To determine biological alterations, gene ontology (GO) and KEGG pathway analyses were implied using the identified DEGs. Hub genes were determined using protein-protein interaction (PPI) network analysis and an interactome was constructed using NetworkAnalyst. Furthermore, five hub genes were evaluated for use as prognostic markers by using the human protein atlas (HPA) and the GEPIA2.0 database. In addition, the correlations between hub-gene expression and immune cell infiltration of oral squamous cell carcinoma (OSCC) tumors were analyzed using the tWumor immune estimation resource (TIMER) database. Results: A total of 2071 upregulated genes and 1893 downregulated genes were identified. GO and pathway analysis showed DEGs were enriched in multiple immune response terms and interaction of inflammatory cytokines. From the PPI network, five hub genes were identified that have a crucial role in OSCC. These included interferon regulatory factor 4 (IRF4), chemokine receptor 7 (CCR7), TNF receptor superfamily member 17 (TNFRSF17), CD27, and sphingosine-1-phosphate receptor 4 (S1PR4), which were predicted to be favorable prognostic markers for OSCC using HPA. Overall survival analysis revealed that low expression of the five hub genes was significantly associated with worse overall survival. Our analysis of tumor-associated immune infiltration revealed that increased IRF4 expression was positively correlated with the gene expression profiles suggestive of infiltration of all immune cell types, whereas increased CCR7 expression was negatively correlated with neutrophil infiltration. Increased expression of CD27, S1PR4, and TNFRSF17 was found to be negatively correlated with dendritic cell, M0 macrophage, and neutrophil infiltration. Conclusion: In summary, inflammation, and the immune response play an important role in OSCC. All five hub genes were good predictors of OSCC prognosis, suggesting that they could be used as potential therapeutic targets and tumor markers.

Histological features of knee osteoarthritis treated with triamcinolone acetonide and hyaluronic acid.

Osteoarthritis (OA) is one of the most common degenerative joint diseases leading to disability in the end stage. Although intra-articular triamcinolone acetonide (TA) is one of the OA treatments that have been widely used, the side effects of such corticosteroids are still controversial. Intra-articular hyaluronic acid (HA) injection is another therapeutic option for patients with OA who do not want to use corticosteroids because of their side effects. However, the difference between the histological features associated with TA and HA in the treatment of OA remains unclear. Thus, the present study aimed to compare the histological effects of TA and HA on the cartilage of patients with knee OA. In the current study, 31 patients diagnosed with grade 3-4 knee OA on the Kellgren-Lawrence radiographic grading scale were separated into three groups: TA (n=12); HA (n=7) and untreated group (n=12). Histological examination of the whole articular cartilages of the patients was performed with hematoxylin and eosin and Alcian staining, as well as using a TUNEL assay. Clinical data such as cartilage thickness, structural and component deterioration, proteoglycan levels, apoptosis and empty lacunae were compared between the three groups. The results showed a high level of deterioration in both TA and HA groups but not in the untreated group, although the thickness of cartilage in the HA group was lower compared with that in the TA and untreated groups. The proteoglycan levels in the TA group were lower compared with those in the HA group. Moreover, the number of empty lacunae in the HA group was higher compared with that in the TA group, while no difference in apoptosis was found between TA and HA groups. A significant difference was not found in the histological staining between TA and HA groups. On the other hand, a significant difference was found in cartilage deterioration between the medial and lateral sides in these groups. TA and HA groups showed comparable histological results. TA injection is cheaper and easier but has more adverse effects for patients with knee OA than HA injection. Therefore, orthopaedists should select TA or HA based on the economic and specific needs of patients.

Rapid decalcification of articular cartilage and subchondral bone using an ultrasonic cleaner with EDTA.

Articular cartilage and subchondral bones were used to be the samples for studying effects of drugs in the joint degenerative diseases such as osteoarthritis. Because of the deposition of mineral salts, articular cartilage and subchondral bones require decalcification process to soften the tissues. EDTA is a chelating agent that is commonly used to remove mineral salts, but this step is time-consuming and can take as long as 45 days. Commercial ultrasonic cleaner and microwave oven were reported to reduce the decalcification timing. The aim of this study is to determine and compare the decalcification of human articular cartilage and subchondral bone using EDTA together with ultrasonic cleaner or microwave oven. Hundred pieces of articular cartilage and subchondral bones obtained from osteoarthritis patients undergone total-knee-replacement were divided into 10 groups according to decalcification method (ultrasonic cleaner or microwave) and timing (2, 4, 6, 8, and 10 h). In each group, all cartilage and subchondral bone pieces were decalcified and sectioned, and subsequently stained with haematoxylin and eosin, Von Kossa, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, or caspase-3 immunohistochemistry. The optimal timing of decalcification of articular cartilage and subchondral bones using EDTA together with ultrasonic cleaner was at 8 and 10 h, while the timing using EDTA together with microwave oven was more than 10 h. Clear TUNEL and caspase-3 signals were obtained from samples decalcified using EDTA together with ultrasonic cleaner for 8 h. In summary, to our knowledge, this is the first study that compared EDTA decalcification between ultrasonic cleaner and microwave oven. Here, we report a new methodology for decalcification for articular cartilage and subchondral bones that reduces decalcification time from weeks to hours and is suitable for further pathological analyses.

Influence of Lithium- and Zinc-Containing Bioactive Glasses on Pulpal Regeneration.

OBJECTIVE: To evaluate the potential of modified bioactive glasses containing lithium and zinc as pulp capping materials by investigating the odontogenic differentiation and mineralization response in the tooth culture model. MATERIALS AND METHODS: Lithium- and zinc-containing bioactive glasses (45S5.1Li, 45S5.5Li, 45S5.1Zn, 45S5.5Zn, 45S5.1Zn sol-gel, and 45S5.5Zn sol-gel), fibrinogen-thrombin, and biodentine were prepared to assess Axin2 gene expression at 0, 30 minutes, 1 hour, 12 hours, and 1 day and DSPP gene expression at 0, 3, 7, and 14 days in stem cells from human exfoliated deciduous teeth (SHEDs) using qRT-PCR. The experimental bioactive glasses incorporated with fibrinogen-thrombin and biodentine were placed on the pulpal tissue in the tooth culture model. Histology and immunohistochemistry were analyzed at 2 weeks and 4 weeks. RESULTS: Axin2 gene expression for all experimental groups was significantly higher than the control at 12 hours. The DSPP gene expression for all experimental groups was significantly higher than the control at 14 days. The presence of mineralization foci was significantly higher at 4 weeks for the modified bioactive glasses 45S5.5Zn, 45S5.1Zn sol-gel, and 45S5.5Zn sol-gel as well as Biodentine compared with the fibrinogen-thrombin control. CONCLUSION: Lithium- and zinc-containing bioactive glasses increased Axin2 and DSPP gene expression in SHEDs and can potentially enhance pulp mineralization and regeneration. Zinc-containing bioactive glasses are a promising candidate to be used as pulp capping materials.

Qualitative Histological Evaluation of Various Decalcifying Agents on Human Dental Tissue.

OBJECTIVE: Dental hard tissue is among the hardest tissue of humans because it contains high amounts of inorganic substances. This leads to difficulty in preparing histological sections for histopathological examination. Acid and chelating agents are generally used to decalcify teeth. We aimed to compare the histological quality of teeth decalcified with various calcifying agents including 5% nitric acid, 50% formic acid with 20% sodium citrate (Anna Morse solution), 10% formic acid, commercial solution, and 14.4% neutral EDTA. MATERIALS AND METHODS: Freshly extracted premolar teeth were fixed and submitted for decalcification using different agents. Histological examination was qualitatively evaluated for tissue integrity and staining quality. RESULTS: Dentin integrity of teeth decalcified with all decalcifying agents did not show any statistical differences except that with the formic acid, whereas cementum integrity decalcified with neutral EDTA showed a superior score compared with other agents. Tissue integrity and staining quality of dental pulp cells were the best decalcified with neutral EDTA or Anna Morse solution. CONCLUSION: Our findings demonstrated that EDTA and Anna Morse solution gave a similar efficiency in the preservation of tissue integrity while Anna Morse solution may be recommended as a decalcification agent in routine use due to the more satisfying decalcification time than EDTA.

Epstein-Barr Virus Detection and LMP-1 Expression in Thai Patients with Oral Squamous Cell Carcinoma.

BACKGROUND: To date, high-risk human papillomavirus (HPV) is primarily linked to oropharyngeal carcinoma, but only a small subset of oral squamous cell carcinoma (OSCC) is truly driven by high-risk HPV. In addition, Epstein-Barr virus (EBV) is another potential oncogenic virus for OSCC development. This study aims to investigate the role of EBV infection in Thai patients with OSCC. METHODS: Forty-seven formalin-fixed paraffin-embedded specimens of OSCC were obtained. EBV DNA was detected by polymerase chain reaction analysis using primers for LMP-1 region of EBV. EBV-positive OSCC cases were subjected to LMP-1 immunohistochemical analysis and EBV-encoded small RNA (EBER) in situ hybridization to determine EBV cellular localization in OSCC. LMP-1 immunohistochemical analysis was also performed in all EBV-negative OSCC cases. RESULTS: Of the 47 OSCC specimens, ten (21%) exhibited EBV DNA by PCR analysis. Seven of ten (70%) EBV-positive specimens showed high-grade LMP-1 expression by immunohistochemistry. However, no EBER expression was detected in all EBV-positive OSCC specimens. In EBV-negative specimens, LMP-1 was also negative except in 3 specimens which showed low grade expression of LMP-1. CONCLUSION: The prevalence of EBV infection in OSCC in this group of Thai patients was 21%. Most of EBV-positive OSCC cases showed LMP-1 expression but a lack of EBER expression. From our findings, we presume that EBV may take some roles in OSCC development in this group of participants.

Effects of Chrysin on Oral Squamous Cell Carcinoma In Vitro.

OBJECTIVE: Chrysin is a hydroxylated flavonoid derived from "propolis or bee glue," a natural product. Previous research on chrysin's biological functions, including anticancer activity, had been reported. However, chrysin's effect on oral squamous cell carcinoma (OSCC) is still scarce. This article aimed to test the cytotoxicity, antiproliferative, antimigration, anti-invasion, and apoptotic effects of purified chrysin in two OSCC cell lines, HSC4 and SCC25. MATERIALS AND METHODS: The malignant phenotype was assessed using cell proliferation, wound healing, and transwell assays. Cell apoptosis was determined using flow cytometry. The positive control was OSCC cells treated with cisplatin, and the negative control was OSCC cells incubated with 0.1% dimethyl sulfoxide. RESULTS: Chrysin at concentrations of 100 and 200 µM could inhibit OSCC cell proliferation, migration, and invasion, as well as enhance cell apoptosis, particularly in the early stages of apoptosis. CONCLUSION: In OSCC cell lines, chrysin has been demonstrated to be an effective antioncogenic agent. Additional research is required to confirm the results. Chrysin should be suggested as a possible alternative therapeutic application for OSCC.