Evaluation of oral brush liquid-based cytology for oral squamous cell carcinoma: a comparative study of cytological and histological diagnoses at a single center.

PURPOSE: Liquid-based cytology is highly useful in oral cytology. However, there are only few reports on the accuracy of this method. The current study aimed to compare oral liquid-based cytological and histological diagnoses and to evaluate items that should be considered in oral cytological diagnosis for oral squamous cell carcinoma. METHODS: We included 653 patients who underwent both oral cytological and histological examinations. Data on sex, specimen collection region, cytological and histological diagnoses, and histological images were reviewed. RESULTS: The overall male-to-female ratio was 1:1.18. The tongue was the most common specimen collection region, followed by the gingiva and buccal mucosa. The most common cytological examination result was negative (66.8%), followed by doubtful (22.7%) and positive (10.3%). The sensitivity, specificity, positive predictive value, and negative predictive value of cytological diagnosis were 69%, 75%, 38%, and 92%, respectively. Approximately 8.3% of patients with a negative cytological diagnosis had a histological diagnosis of oral squamous cell carcinoma. Furthermore, 86.1% of histopathologic images of cytology-negative squamous cell carcinomas exhibited well-differentiated keratinocytes lacking atypia on the surface. The remaining patients developed recurrence, or they had low cell counts. CONCLUSION: Liquid-based cytology is useful in screening oral cancer. However, a cytological diagnosis of superficial-differentiated oral squamous cell carcinoma is occasionally inconsistent with the histological diagnosis. Therefore, histological and cytological examinations should be performed if tumor-like lesions are suspected clinically.

Congenital Epulis: A Case and Review of the Literature.

Congenital epulis is an unusual benign oral mucosal lesion in newborns with no tendency to recur after excision. The histogenesis of the lesion is unknown, but it is believed to be of mesenchymal origin. We describe a case of congenital epulis (20×10 mm) in the mandibular gingiva of a newborn. The mass, which was smooth-surfaced and pedunculated with a healthy color, was surgically removed at 5 months post-birth. Histologically, the tumor consisted mainly of large eosinophilic granular cells. Immunohistochemical studies revealed intense staining for vimentin, STRO-1, and CD44, suggesting that it was derived from mesenchymal cells. The literature and immunohistochemical profile of congenital epulis are also discussed.

Clinicopathological evaluation of oral leukoplakia: a single-center study of 676 cases in Japan.

OBJECTIVES: To analyze the clinical and histopathological characteristics of oral leukoplakia (OL) in the Japanese population and investigate the prevalence and risk factors for epithelial dysplasia (ED) and carcinoma within lesions. STUDY DESIGN: Data, including age, sex, lesion site, and histopathological features, of 676 cases diagnosed with OL over the previous 10 years were analyzed. Dysplasia and carcinoma prevalence were determined. RESULTS: In male patients, the most affected site was the gingiva (42.7%), whereas in females, it was the tongue (47.6%). Moreover, ED was more prevalent in males (41.9%), whereas epithelial hyperplasia was more common in females (44.7%). A significant difference was observed between affected sites with regard to the presence of dysplasia. The ED rates by site were 64.6% and 33.7% for the tongue and gingiva, respectively (P < 0.05). The squamous cell carcinoma rates by site were 23.4%, 5.4%, and 3.4% for the tongue, buccal mucosa, and gingiva, respectively (P < 0.05). Logistic regression analysis revealed a higher prevalence of dysplasia in males than it did in females and that the risk for both dysplasia and carcinoma was highest in the tongue. CONCLUSIONS: Dysplasia is common in OL cases, often showing carcinoma. Early biopsy and interventions are key in OL management.

The Effects of Tricalcium-Silicate-Nanoparticle-Containing Cement: In Vitro and In Vivo Studies.

A tricalcium-silicate-nanoparticle-containing cement (Biodentine) was developed to overcome the disadvantages of existing mineral trioxide aggregate (MTA) dental materials. This study aimed at evaluating the effects of Biodentine on the osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs) in vitro and the healing of furcal perforations created experimentally in rat molars in vivo, in comparison to MTA. The in vitro studies performed the following assays: pH measurement using a pH meter, the release of calcium ions using a calcium assay kit, cell attachment and morphology using SEM, cell proliferation using a coulter counter, marker expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and cell mineralized deposit formation using Alizarin Red S (ARS) staining. In the in vivo studies, MTA and Biodentine were used to fill the rat molar perforations. Rat molars were processed at 7, 14 and 28 days for analysis of inflammatory processes using hematoxylin and eosin (HE) staining, immunohistochemical staining of Runx2 and tartrate-resistant acid phosphate (TRAP) staining. The results demonstrate that the nanoparticle size distribution of Biodentine is critical for osteogenic potential at an earlier stage compared to MTA. Further studies are required to elucidate the mechanism of action of Biodentine in osteogenic differentiation.

Intraoral Minor Salivary Gland Tumors: A Retrospective, Clinicopathologic, Single-Center Study of 432 Cases in Japan and a Comparison with Epidemiological Data.

BACKGROUND: Intraoral minor salivary gland tumors are relatively rare lesions with histological subtypes not commonly found in major salivary glands. This study aimed to retrospectively evaluate the clinicopathologic features of intraoral minor salivary gland tumors from the Tokyo Dental College Hospital, Japan, and compare them with findings from other epidemiological studies. METHODS: We conducted a retrospective clinicopathologic evaluation of 432 cases of intraoral minor salivary gland tumors [161 male (37.3%) and 271 female (62.7%) patients; mean age: 52.5 and 48.6 years for males and females, respectively; age at diagnosis: 7-87 (mean: 50.1) years] from the Tokyo Dental College Hospital between 1975 and 2022, including 283 benign tumors (65.5%) and 149 malignant tumors (34.5%). RESULTS: The most common benign tumor was pleomorphic adenoma (n = 239), whereas mucoepidermoid carcinoma was the most common malignant tumor (n = 74). The mean age of patients with benign and malignant tumors was 48.4 and 53.2 years, respectively, with patients with malignant tumors being significantly older (P = 0.0042). The mean age of patients with malignant tumors was significantly higher in males (56.7 years) than in females (50.9 years) (P = 0.0376), although the mean age of patients with benign tumors did not differ by sex. Tumors were commonly located in the palate [250 cases (57.9%)]. Benign tumors were more frequent in the palate, upper lip, and buccal mucosa, whereas malignant tumors were more frequent in the palate, floor of the mouth, buccal mucosa, and retromolar area. CONCLUSIONS: Understanding the features of intraoral minor salivary gland tumors is useful for diagnosis. Our study provides important epidemiological data (patient differences in age at occurrence, sex, and site of origin) that will inform clinicians and researchers.

Genetic Study of BRAF V600E and SMO L412F Mutations in Japanese Patients with Ameloblastoma.

Background and aim. Ameloblastoma is a benign, intraosseous, progressively growing, epithelial, odontogenic neoplasm. BRAF and SMO mutations have been reported in ameloblastoma. In this study, we evaluated BRAF V600E and SMO L412F mutations; and assessed the relationship between BRAF V600E mutant expression and the clinicopathological features in Japanese patients with ameloblastoma. Methods. We examined 24 formalin-fixed paraffin-embedded samples. All specimens were from patients with mandibular ameloblastoma: 20 were conventional ameloblastoma and 4 were unicystic ameloblastoma. The BRAF V600E mutation was assessed by Sanger sequencing and immunohistochemistry, and the SMO L412F mutation was assessed only by Sanger sequencing. Results. Twenty of the 24 (83%) ameloblastoma samples carried the BRAF V600E mutation; 22 of the 24 (92%) samples were immunohistochemically positive for BRAF V600E. However, the SMO L412F mutation was not detected in any of them. The BRAF V600E mutation status did not correlate with the clinicopathological features, such as age, sex, location, method, recurrence, and subtype. Conclusion. BRAF inhibitors could be a potential treatment option for Japanese patients with ameloblastoma, harboring the BRAF V600E mutation.

Frequency of Odontogenic Tumors: A Single Center Study of 1089 Cases in Japan and Literature Review.

Several attempts have been made to classify odontogenic tumors; however, the need for a uniform international classification system led the World Health Organization (WHO) to present a classification of odontogenic tumors in 1971. We aimed to evaluate the number and types of odontogenic tumors examined at the Tokyo Dental College Hospital in Japan to determine the frequency and types of odontogenic tumors, based on the 2017 WHO classification system, as this information has not been reported previously in Japan. We also compared the results of our evaluation with those reported in previous studies. We conducted a clinicopathological evaluation of odontogenic tumors examined at the Tokyo Dental College Hospital between 1975 and 2020. This included an analysis of 1089 cases (malignant, n = 10, 0.9%; benign, n = 1079, 99.1%) based on the 2017 World Health Organization Classification of Head and Neck Tumors. We identified 483 (44.3%), 487 (44.7%), and 109 (10.0%) benign epithelial odontogenic, mixed odontogenic, and mesenchymal tumors, respectively. The most common tumor types were odontoma (42.5%) and ameloblastoma (41.9%). Of the 1089 cases, 585 (53.7%) and 504 (46.3%) were male and female patients, respectively. Ameloblastoma and ameloblastic fibroma occurred more commonly in male patients, whereas odontogenic fibroma and cemento-ossifying fibroma affected female patients primarily. The age at diagnosis ranged from three to 87 (mean, 29.05) years. In 319 (29.3%) patients, the age at diagnosis ranged from 10 to 19 years. Ameloblastoma and odontoma were the most common tumor types among patients in their 20s and those aged 10-19 years, respectively. In 737 (67.7%) and 726 (66.7%) patients, the tumors were located in the mandible and posterior region, respectively. Ameloblastoma was particularly prevalent in the posterior mandible. Odontogenic tumors are rare lesions and appear to show a definite geographic variation.

Effects of Excimer Laser Treatment of Zirconia Disks on the Adhesion of L929 Fibroblasts.

The adhesion of zirconia and soft tissue is very important for the success of zirconia implants. The aim of this study was to characterize the effects of excimer laser treatment of zirconia on the adhesion of L929 fibroblasts. In this study, polished zirconia disks treated with an excimer laser were the experimental group and untreated zirconia disks were the control group. Surface roughness and contact angles of zirconia disks were measured. mRNA expression levels of integrin ß1 and collagen type I α1 in L929 fibroblasts cultured on zirconia disks were measured using qRT-PCR. Cell morphology was evaluated using 3D laser microscopy and the expression of vinculin was characterized using confocal microscopy. There was no significant difference in the surface roughness of zirconia disks, but contact angles were significantly lower. mRNA expression of integrin ß1 was significantly higher at 3, 6 and 24 h and of collagen type I α1 was significantly higher at 6 and 24 h. L929 fibroblasts tended to form elongated microspikes and vinculin colocalization in those microspikes. Furthermore, vinculin was strongly expressed in filopodia of L929 fibroblasts at 24 h. These results suggest that excimer laser treatment improves adhesion between zirconia disks and L929 fibroblasts.

The effect of fibroblast growth factor 7 on human dental pulp stem cells for differentiation to AQP5-positive and αSMA-positive cells in vitro and in vivo.

OBJECTIVES: Transplantation of stem cells into wounds has become popular in regeneration therapies. As stem cells for transplantation, human dental pulp stem cells (hDPSCs) are known to be pluripotent cells that are relatively easy to collect from the pulp of deciduous or wisdom teeth. The purpose of this study was to investigate whether hDPSCs treated with fibroblast growth factor 7 (FGF7) would contribute to the regeneration of wounded rat submandibular glands (SMGs). MATERIALS AND METHODS: In in vitro studies, hDPSCs were treated with or without FGF7 and mRNA expression levels were examined at days 3, 7 and 14 using qRT-PCR. The target genes analyzed were BMI1 as an undifferentiated marker, AQP5 as an acinar cell marker, CK19 as a ductal epithelial cell marker, αSMA as a myoepithelial cell marker and VIMENTIN as a fibroblast marker. In in vivo studies, hDPSCs treated with or without FGF7 for 14 days were mixed with type I collagen gels and were transplanted into wounded rat SMGs. Hematoxylin-Eosin and immunohistochemical staining were performed at days 3 and 7, and the numbers of positive cells were counted. The primary antibodies used were against BMI1, AQP5, αSMA, PanCK and VIMENTIN. RESULTS: In the in vitro studies, mRNA levels of BMI1 were decreased and αSMA were increased at days 3, 7 and 14, while AQP5 was increased at day 14 in the FGF7 group. In the in vivo studies, the proliferation of hDPSCs and cell islands was observed at day 7 in the FGF7 group. Few BMI1-positive cells were observed, while numbers of AQP5-positive and αSMA-positive cells were increased at days 3 and 7 in the FGF7 group. Moreover, cell islands were AQP5-positive. CONCLUSION: These results suggest that FGF7-treated hDPSCs differentiate into AQP5-positive and αSMA-positive cells. Moreover, AQP5-positive cell aggregations were formed.

Motif-programmed artificial extracellular matrix.

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.

Immobilization of a carbon nanomaterial-based localized drug-release system using a bispecific material-binding peptide.

INTRODUCTION: Inorganic materials are widely used in medical devices, such as artificial hearts, vessels, and joints, in stents, and as nanocarriers for drug-delivery systems. Carbon nanomaterials are of particular interest due to their biological inertness and their capability to accommodate molecules. Several attempts have been proposed, in which carbon nanomaterials are used as nanocarriers for the systemic delivery of drugs. MATERIALS AND METHODS: We developed a drug-delivery system in which oxidized single-walled carbon nanohorns (oxSWNHs) were immobilized on a titanium (Ti) surface using material-binding peptides to enable localized drug delivery. For this purpose, we utilized a bispecific peptidic aptamer comprising a core sequence of a Ti-binding peptide and a SWNH-binding peptide to immobilize oxSWNHs on Ti. RESULTS: Scanning electron microscopy was used to confirm the presence of oxSWNHs adsorbed onto the Ti surface, and a quartz crystal microbalance was used to evaluate the binding process during oxSWNH adsorption. The oxSWNHs-ornamented Ti substrate was nontoxic to cells and released biologically active dexamethasone over a sustained period. CONCLUSION: This oxSWNHs-immobilized system can be used to modify the surface of Ti in implants and be loaded with drugs that stimulate osteogenesis and bone regeneration.

Profiling of circulating exosomal miRNAs in patients with Waldenström Macroglobulinemia.

Waldenström Macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by disease progression from IgM MGUS to asymptomatic and then symptomatic disease states. We profiled exosomes from the peripheral blood of patients with WM at different stages (30 smoldering/asymptomatic WM, 44 symptomatic WM samples and 10 healthy controls) to define their role as potential biomarkers of disease progression. In this study, we showed that circulating exosomes and their miRNA content represent unique markers of the tumor and its microenvironment. We observed similar levels of miRNAs in exosomes from patients with asymptomatic (smoldering) and symptomatic WM, suggesting that environmental and clonal changes occur in patients at early stages of disease progression before symptoms occur. Moreover, we identified a small group of miRNAs whose expression correlated directly or inversely with the disease status of patients, notably the known tumor suppressor miRNAs let-7d and the oncogene miR-21 as well as miR-192 and miR-320b. The study of these miRNAs' specific effect in WM cells could help us gain further insights on the mechanisms underlying WM pathogenesis and reveal their potential as novel therapeutic targets for this disease.

Platelets Enhance Multiple Myeloma Progression via IL-1ß Upregulation.

Purpose: Tumor cell-platelet interactions contribute to tumor progression and metastasis in solid tumors. However, the role of platelets in hematological malignancies is not clear. We investigated the association of platelet activation status with clinical stages in multiple myeloma (MM) patients and explored the role of platelets in MM progression.Experimental Design: Platelets were obtained from healthy donors and MM patients. We examined platelet activation status in MM patients by flow cytometry and transmission electron microscopy. We also observed the enriched pathways that are involved with platelet activation in RNA sequencing of platelets. MM cell lines were used to assess the effect of platelets on MM cell proliferation in vitro and their engraftment in vivo RNA sequencing of MM cell lines was performed to explore molecular mechanisms underlying MM cell-platelet interaction and a CRISPR/Cas9 knockout approach was used for validation.Results: Platelets from MM patients were highly activated with disease progression. RNA sequencing of platelets revealed that genes involved in platelets were enriched in patients with smoldering MM (SMM) or MM. Platelets promoted MM cell proliferation in vitro and contributed to tumor engraftment in bone marrow in vivo RNA sequencing revealed that IL-1ß was upregulated in MM cell lines co-cultured with platelets, whereas IL-1ß knockout in MM cell lines abrogated the effects of platelets on MM cell proliferation and engraftment in vivoConclusions: Platelets from MM patients were highly activated with disease progression. IL-1ß is critical to platelet-mediated MM progression and might be a potential target for MM treatment. Clin Cancer Res; 24(10); 2430-9. ©2018 AACR.

Inhibition of microRNA-138 enhances bone formation in multiple myeloma bone marrow niche.

Myeloma bone disease is a devastating complication of multiple myeloma (MM) and is caused by dysregulation of bone remodeling processes in the bone marrow microenvironment. Previous studies showed that microRNA-138 (miR-138) is a negative regulator of osteogenic differentiation of mesenchymal stromal cells (MSCs) and that inhibiting its function enhances bone formation in vitro. In this study, we explored the role of miR-138 in myeloma bone disease and evaluated the potential of systemically delivered locked nucleic acid (LNA)-modified anti-miR-138 oligonucleotides in suppressing myeloma bone disease. We showed that expression of miR-138 was significantly increased in MSCs from MM patients (MM-MSCs) and myeloma cells compared to those from healthy subjects. Furthermore, inhibition of miR-138 resulted in enhanced osteogenic differentiation of MM-MSCs in vitro and increased the number of endosteal osteoblastic lineage cells (OBCs) and bone formation rate in mouse models of myeloma bone disease. RNA sequencing of the OBCs identified TRPS1 and SULF2 as potential miR-138 targets that were de-repressed in anti-miR-138-treated mice. In summary, these data indicate that inhibition of miR-138 enhances bone formation in MM and that pharmacological inhibition of miR-138 could represent a new therapeutic strategy for treatment of myeloma bone disease.

Blocking IFNAR1 inhibits multiple myeloma-driven Treg expansion and immunosuppression.

Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression is the expansion of regulatory T cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type 1 interferon (IFN). Blocking IFN α and ß receptor 1 (IFNAR1) on Tregs significantly decreases both myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of MM patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type 1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1-knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type 1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

An artificial fusion protein between bone morphogenetic protein 2 and titanium-binding peptide is functional in vivo.

The purpose of this study was to investigate osteogenesis using an artificial fusion protein (AFP) composed of modified bone morphogenetic protein 2 (BMP-2) with a titanium (Ti)-binding peptide (TBP) motif on a Ti surface in vivo. In the in vivostudy, 5-µm thick Ti was coated with electron cyclotron resonance sputtering on a porous carbon scaffold which was then dipped in one of three different mixtures of collagen gel: (1) collagen gel only, (2) collagen gel with TBP, and (3) collagen gel with the AFP between BMP-2 and the TBP motif (AFP-TBP-BMP-2). These scaffolds were then implanted into rat abdominal muscles and were studied histologically at various times and the expression of several bone-related protein messenger RNAs (mRNAs) was also analyzed. The Ti-coated scaffold of the collagen gel with AFP-TBP-BMP-2 produced cartilage in the muscle and the expression of alkaline phosphatase, bone sialoprotein, and runt-related gene 2 mRNAs was significantly increased. These results suggest that the scaffold of the collagen gel with AFP-TBP-BMP-2 accelerates osteogenesis in vivo.

Change in surface properties of zirconia and initial attachment of osteoblastlike cells with hydrophilic treatment.

The objectives of this study were to characterize change in surface properties of tetragonal zirconia polycrystals (TZP) after hydrophilic treatment, and to determine the effect of such changes on initial attachment of osteoblast-like cells. Roughened surfaces were produced by alumina-blasting and acid-etching. Hydrophilic treatment comprised application of immediately after blasting and acid-etching (Blast/Etch), oxygen plasma (O2-Plasma), ultraviolet light (UV). Specimens stored in air were used as a control. The water contact angle was determined and surface analysis was performed using an X-ray photoelectron spectroscopy. Blast/Etch, O2-Plasma and UV specimens showed superhydrophilicity, and these hydrophilic treatments to TZP elicited a marked decrease in carbon content and an increase in hydroxyl groups. Hydrophilic treatments enhanced initial attachment of osteoblast-like cells and a change in cell morphologies. These results indicate that Blast/Etch, O2-Plasma, or UV treatment has potential in the creation and maintenance of superhydrophilic surfaces and enhancing initial attachment of osteoblast-like cells.