Transcutaneous carbon dioxide suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Cancer cachexia causes skeletal muscle atrophy, impacting the treatment and prognosis of patients with advanced cancer, but no treatment has yet been established to control cancer cachexia. We demonstrated that transcutaneous application of carbon dioxide (CO2) could improve local blood flow and reduce skeletal muscle atrophy in a fracture model. However, the effects of transcutaneous application of CO2 in cancer-bearing conditions are not yet known. In this study, we calculated fat-free body mass (FFM), defined as the skeletal muscle mass, and evaluated the expression of muscle atrophy markers and uncoupling protein markers as well as the cross-sectional area (CSA) to investigate whether transcutaneous application of CO2 to skeletal muscle could suppress skeletal muscle atrophy in cancer-bearing mice. Human oral squamous cell carcinoma was transplanted subcutaneously into the upper dorsal region of nude mice, and 1 week later, CO2 gas was applied to the legs twice a week for 4 weeks and FFM was calculated by bioimpedance spectroscopy. After the experiment concluded, the quadriceps were extracted, and muscle atrophy markers (muscle atrophy F-box protein (MAFbx), muscle RING-finger protein 1 (MuRF-1)) and uncoupling protein markers (uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3)) were evaluated by real-time polymerase chain reaction and immunohistochemical staining, and CSA by hematoxylin and eosin staining. The CO2-treated group exhibited significant mRNA and protein expression inhibition of the four markers. Furthermore, immunohistochemical staining showed decreased MAFbx, MuRF-1, UCP2, and UCP3 in the CO2-treated group. In fact, the CSA in hematoxylin and eosin staining and the FFM revealed significant suppression of skeletal muscle atrophy in the CO2-treated group. We suggest that transcutaneous application of CO2 to skeletal muscle suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Squamous cell carcinoma initially occurring on the tongue dorsum: a case series report with molecular analysis.

BACKGROUND: Squamous cell carcinoma (SCC) of the dorsum of the tongue is extremely rare, and it clinically resembles various benign lesions. Somatic mutations in TP53 and some driver genes were implicated in the development of SCC; however, the somatic genetic characteristics of dorsal tongue SCC remain unknown. With a detailed analysis of gene mutations in dorsal tongue SCC, we aimed to better understand its biology. METHODS: Four cases of SCC initially occurring on the tongue dorsum were evaluated for clinical and histological findings and immunohistochemical expression of p53 and p16. Gene mutations were analyzed using next-generation sequencing with a custom panel of driver genes. RESULTS: We retrospectively investigated 557 cases of tongue SCC, and only four cases of SCC initially occurred on the tongue dorsum. The four patients (cases 1-4) were one woman and three men with a mean age of 53.75 years (range: 15-74 years). Histological analysis revealed well-differentiated SCC. Through molecular analysis, we identified pathogenic somatic mutations, namely, TP53 p.C176F (c.527G > T) in case 3 and TP53 p.R282W (c.844 C > T) in case 4. No pathogenic variants were identified in the PI3K/AKT or RAS/RAF pathways. The p53 immunohistochemical examination revealed a wild-type expression pattern in cases 1-3 and strong expression in case 4. The results of p16 immunostaining were negative in all cases. CONCLUSIONS: We described four previously unreported genetic characteristics of dorsal tongue SCC. Somatic TP53 mutations may contribute to the development of a subset of dorsal tongue SCC; however, more cases with genetic analysis need to be accumulated.

Risk factors for post-extraction infection of mandibular third molar: A retrospective clinical study.

Post-extraction infection is one of the most concerning complications of mandibular third molar extraction, which is the most common procedure in oral and maxillofacial surgery. We investigated risk factors for post-extraction infection by retrospectively analyzing 2,513 teeth/cases of mandibular third molar extraction (1,040 males, 1,473 females) performed at a single medical facility in Kobe, Japan from January 2014 to May 2022. The predictive variables were categorized as patient attributes, health status, and anatomic, pathological, and operative variables that may be associated with post-extraction infection. The outcome variable was the post-extraction infection rate. The post-extraction infection rate was 5.73 % (144 of the 2,513 teeth), and the mean age of the patients with a post-extraction infection was 41.76 ± 16.8 years. Our analyses also revealed that the postoperative infection rate was significantly increased in patients aged ≥36 years. A multivariate logistic regression analysis showed that the following variables were significantly associated with post-extraction infection: preoperative antibiotic administration (odds ratio [OR] 4.68, p < 0.001), postoperative paresthesia of the inferior alveolar nerve (OR 4.34, p < 0.001), intraoperative hemostatic procedure (OR 1. 74, p = 0.008), position of Pell and Gregory classifications (OR 1. 70, p < 0.001), Winter's classification (OR 1.28, p < 0.03), and age (OR 1.03, p < 0.001). Oral and maxillofacial surgeons should be aware of these risk factors.

Novel mechanism of cisplatin resistance in head and neck squamous cell carcinoma involving extracellular vesicles and a copper transporter system.

BACKGROUND: Cisplatin (CDDP) plays a central role in chemotherapy for head and neck squamous cell carcinoma (HNSCC), but drug resistance in HNSCC chemotherapy remains a problem, and the mechanism of CDDP resistance is unclear. We investigated CDDP-resistance mechanisms mediated by extracellular vesicles (EVs) and ATPase copper transporting beta (ATP7B) in HNSCC. METHODS: We established CDDP-resistant sublines of HNSCC cells and verified their ATP7B expression. We used an EV secretion inhibitor (GW4869) and ATP7B short hairpin (sh)RNA transfection to examine the correlation between EV secretion and ATP7B expression. RESULTS: The CDDP-resistant HNSCC sublines showed decreased CDDP sensitivity and increased ATP7B expression. GW4869 suppressed ATP7B expression, and ATP7B shRNA transfection suppressed EV secretion. The suppressions of EV secretion and ATP7B expression both enhanced CDDP's cell-killing effect. CONCLUSIONS: EVs were involved in the ATP7B-mediated mechanism underlying CDDP resistance. Further clarification of the EV-induced CDDP-resistance mechanism may lead to novel therapeutic strategies for HNSCC.

Rab11 suppresses head and neck carcinoma by regulating EGFR and EpCAM exosome secretion.

OBJECTIVES: Rab11(Rab11a and Rab11b) localizes primarily along recycling endosomes in cells and is involved in various intracellular trafficking processes, including membrane receptor recycling and secretion of exosomes or small extracellular vesicles (EVs). Although Rab11 is closely associated with the progression and metastasis of various cancer types, little is known about Rab11' role in head and neck squamous cell carcinoma (HNSCC). In this study, we investigated the roles of Rab11a and Rab11b in HNSCC. METHODS: The clinical significance of Rab11 expression in HNSCC was investigated using a public database and tissue microarray analysis. Stable cell lines with loss and gain of Rab11a or Rab11b were originally established to investigate their roles in the proliferative, migratory, and invasive capabilities of HNSCC cells. RESULTS: Database analysis revealed a significant association between Rab11b mRNA expression and a favorable patient survival rate in HNSCC. Tissue microarray analysis revealed that Rab11b expression was the highest in normal tissues and gradually decreased across the stages of HNSCC progression. Overexpression of Rab11a or Rab11b resulted in a decrease in epidermal growth factor receptor (EGFR), Epithelial cell adhesion molecule (EpCAM) exosome secretion, and the migratory and invasive potential of HNSCC cells. The knockdown of Rab11a or Rab11b increased EpCAM/CD9 exosome secretion in addition to the migratory and invasive potential of HNSCC cells. CONCLUSIONS: Rab11 suppresses HNSCC by regulating EGFR recycling and EpCAM exosome secretion in HNSCC cells. Our results indicate that Rab11b is a superior prognostic indicator of HNSCC and holds promise for developing novel therapeutic strategies.

Ligneous periodontitis exacerbated by Behçet's disease in a patient with plasminogen deficiency and a stop-gained variant PLG c.1468C > T: a case report.

BACKGROUND: Plasminogen serves as the precursor to plasmin, an essential element in the fibrinolytic process, and is synthesized primarily in the liver. Plasminogen activation occurs through the action of plasminogen activator, converting it into plasmin. This conversion greatly enhances the fibrinolytic system within tissues and blood vessels, facilitating the dissolution of fibrin clots. Consequently, congenital deficiency of plasminogen results in impaired fibrin degradation. Patients with plasminogen deficiency typically exhibit fibrin deposits in various mucosal sites throughout the body, including the oral cavity, eyes, vagina, and digestive organs. Behcet's disease is a chronic recurrent systemic inflammatory disease with four main symptoms: aphthous ulcers of the oral mucosa, vulvar ulcers, skin symptoms, and eye symptoms, and has been reported worldwide. This disease is highly prevalent around the Silk Road from the Mediterranean to East Asia. We report a case of periodontitis in a patient with these two rare diseases that worsened quickly, leading to alveolar bone destruction. Genetic testing revealed a novel variant characterized by a stop-gain mutation, which may be a previously unidentified etiologic gene associated with decreased plasminogen activity. CASE PRESENTATION: This case report depicts a patient diagnosed with ligneous gingivitis during childhood, originating from plasminogen deficiency and progressing to periodontitis. Genetic testing revealed a suspected association with the PLG c.1468C > T (p.Arg490*) stop-gain mutation. The patient's periodontal condition remained stable with brief intervals of supportive periodontal therapy. However, the emergence of Behçet's disease induced acute systemic inflammation, necessitating hospitalization and treatment with steroids. During hospitalization, the dental approach focused on maintaining oral hygiene and alleviating contact-related pain. The patient's overall health improved with inpatient care and the periodontal tissues deteriorated. CONCLUSIONS: Collaborative efforts between medical and dental professionals are paramount in comprehensively evaluating and treating patients with intricate complications from rare diseases. Furthermore, the PLG c.1468C > T (p.Arg490*) stop-gain mutation could contribute to the association between plasminogen deficiency and related conditions.

Effectiveness of deep learning classifiers in histopathological diagnosis of oral squamous cell carcinoma by pathologists.

The study aims to identify histological classifiers from histopathological images of oral squamous cell carcinoma using convolutional neural network (CNN) deep learning models and shows how the results can improve diagnosis. Histopathological samples of oral squamous cell carcinoma were prepared by oral pathologists. Images were divided into tiles on a virtual slide, and labels (squamous cell carcinoma, normal, and others) were applied. VGG16 and ResNet50 with the optimizers stochastic gradient descent with momentum and spectral angle mapper (SAM) were used, with and without a learning rate scheduler. The conditions for achieving good CNN performances were identified by examining performance metrics. We used ROCAUC to statistically evaluate diagnostic performance improvement of six oral pathologists using the results from the selected CNN model for assisted diagnosis. VGG16 with SAM showed the best performance, with accuracy = 0.8622 and AUC = 0.9602. The diagnostic performances of the oral pathologists statistically significantly improved when the diagnostic results of the deep learning model were used as supplementary diagnoses (p-value = 0.031). By considering the learning results of deep learning model classifiers, the diagnostic accuracy of pathologists can be improved. This study contributes to the application of highly reliable deep learning models for oral pathological diagnosis.

The Role of Hedgehog Signaling in the Melanoma Tumor Bone Microenvironment.

A crucial regulator in melanoma progression and treatment resistance is tumor microenvironments, and Hedgehog (Hh) signals activated in a tumor bone microenvironment are a potential new therapeutic target. The mechanism of bone destruction by melanomas involving Hh/Gli signaling in such a tumor microenvironment is unknown. Here, we analyzed surgically resected oral malignant melanoma specimens and observed that Sonic Hedgehog, Gli1, and Gli2 were highly expressed in tumor cells, vasculatures, and osteoclasts. We established a tumor bone destruction mouse model by inoculating B16 cells into the bone marrow space of the right tibial metaphysis of 5-week-old female C57BL mice. An intraperitoneal administration of GANT61 (40 mg/kg), a small-molecule inhibitor of Gli1 and Gli2, resulted in significant inhibition of cortical bone destruction, TRAP-positive osteoclasts within the cortical bone, and endomucin-positive tumor vessels. The gene set enrichment analysis suggested that genes involved in apoptosis, angiogenesis, and the PD-L1 expression pathway in cancer were significantly altered by the GANT61 treatment. A flow cytometry analysis revealed that PD-L1 expression was significantly decreased in cells in which late apoptosis was induced by the GANT61 treatment. These results suggest that molecular targeting of Gli1 and Gli2 may release immunosuppression of the tumor bone microenvironment through normalization of abnormal angiogenesis and bone remodeling in advanced melanoma with jaw bone invasion.

EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma.

OBJECTIVES: Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance. MATERIALS AND METHODS: We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS). RESULTS: EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner. CONCLUSION: Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.

Enzyme-Cleaved Bone Marrow Transplantation Improves the Engraftment of Bone Marrow Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC) therapy is a promising approach to curing bone diseases and disorders. In treating genetic bone disorders, MSC therapy is local or systemic transplantation of isolated and in vitro proliferated MSC rather than bone marrow transplantation. Recent evidence showed that bone marrow MSC engraftment to bone regeneration has been controversial in animal and human studies. Here, our modified bone marrow transplantation (BMT) method solved this problem. Like routine BMT, our modified method involves three steps: (i) isolation of bone marrow cells from the donor, (ii) whole-body lethal irradiation to the recipient, and (iii) injection of isolated bone marrow cells into irradiated recipient mice via the tail vein. The significant modification is imported at the bone marrow isolation step. While the bone marrow cells are flushed out from the bone marrow with the medium in routine BMT, we applied the enzymes' (collagenase type 4 and dispase) integrated medium to wash out the bone marrow cells. Then, cells were incubated in enzyme integrated solution at 37°C for 10 minutes. This modification designated BMT as collagenase-integrated BMT (c-BMT). Notably, successful engraftment of bone marrow MSC to the new bone formation, such as osteoblasts and chondrocytes, occurs in c-BMT mice, whereas routine BMT mice do not recruit bone marrow MSC. Indeed, flow cytometry data showed that c-BMT includes a higher proportion of LepR+, CD51+, or RUNX2+ non-hematopoietic cells than BMT. These findings suggested that c-BMT is a time-efficient and more reliable technique that ensures the disaggregation and collection of bone marrow stem cells and engraftment of bone marrow MSC to the recipient. Hence, we proposed that c-BMT might be a promising approach to curing genetic bone disorders. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Novel Artificial Scaffold for Bone Marrow Regeneration: Honeycomb Tricalcium Phosphate.

Bone marrow is complex structure containing heterogenetic cells, making it difficult to regenerate using artificial scaffolds. In a previous study, we succeeded in developing honeycomb tricalcium phosphate (TCP), which is a cylindrical scaffold with a honeycomb arrangement of straight pores, and we demonstrated that TCP with 300 and 500 µm pore diameters (300TCP and 500TCP) induced bone marrow structure within the pores. In this study, we examined the optimal scaffold structure for bone marrow with homeostatic bone metabolism using honeycomb TCP. 300TCP and 500TCP were transplanted into rat muscle, and bone marrow formation was histologically assessed. Immunohistochemistry for CD45, CD34, Runt-related transcription factor 2 (Runx2), c-kit single staining, Runx2/N-cadherin, and c-kit/Tie-2 double staining was performed. The area of bone marrow structure, which includes CD45(+) round-shaped hematopoietic cells and CD34(+) sinusoidal vessels, was larger in 300TCP than in 500TCP. Additionally, Runx2(+) osteoblasts and c-kit(+) hematopoietic stem cells were observed on the surface of bone tissue formed within TCP. Among Runx2(+) osteoblasts, spindle-shaped N-cadherin(+) cells existed in association with c-kit(+)Tie-2(+) hematopoietic stem cells on the bone tissue formed within TCP, which formed a hematopoietic stem cell niche similar to as in vivo. Therefore, honeycomb TCP with 300 µm pore diameters may be an artificial scaffold with an optimal geometric structure as a scaffold for bone marrow formation.

Polymeric Materials, Advances and Applications in Tissue Engineering: A Review.

Tissue Engineering (TE) is an interdisciplinary field that encompasses materials science in combination with biological and engineering sciences. In recent years, an increase in the demand for therapeutic strategies for improving quality of life has necessitated innovative approaches to designing intelligent biomaterials aimed at the regeneration of tissues and organs. Polymeric porous scaffolds play a critical role in TE strategies for providing a favorable environment for tissue restoration and establishing the interaction of the biomaterial with cells and inducing substances. This article reviewed the various polymeric scaffold materials and their production techniques, as well as the basic elements and principles of TE. Several interesting strategies in eight main TE application areas of epithelial, bone, uterine, vascular, nerve, cartilaginous, cardiac, and urinary tissue were included with the aim of learning about current approaches in TE. Different polymer-based medical devices approved for use in clinical trials and a wide variety of polymeric biomaterials are currently available as commercial products. However, there still are obstacles that limit the clinical translation of TE implants for use wide in humans, and much research work is still needed in the field of regenerative medicine.

SOD3 Expression in Tumor Stroma Provides the Tumor Vessel Maturity in Oral Squamous Cell Carcinoma.

Tumor angiogenesis is one of the hallmarks of solid tumor development. The progressive tumor cells produce the angiogenic factors and promote tumor angiogenesis. However, how the tumor stromal cells influence tumor vascularization is still unclear. In the present study, we evaluated the effects of oral squamous cell carcinoma (OSCC) stromal cells on tumor vascularization. The tumor stromal cells were isolated from two OSCC patients with different subtypes: low invasive verrucous squamous carcinoma (VSCC) and highly invasive squamous cell carcinoma (SCC) and co-xenografted with the human OSCC cell line (HSC-2) on nude mice. In comparison, the CD34+ vessels in HSC-2+VSCC were larger than in HSC-2+SCC. Interestingly, the vessels in the HSC-2+VSCC expressed vascular endothelial cadherin (VE-cadherin), indicating well-formed vascularization. Our microarray data revealed that the expression of extracellular superoxide dismutase, SOD3 mRNA is higher in VSCC stromal cells than in SCC stromal cells. Moreover, we observed that SOD3 colocalized with VE-cadherin on endothelial cells of low invasive stroma xenograft. These data suggested that SOD3 expression in stromal cells may potentially regulate tumor vascularization in OSCC. Thus, our study suggests the potential interest in SOD3-related vascular integrity for a better OSCC therapeutic strategy.

Lip pleomorphic adenomas: case series and literature review.

Background: Pleomorphic adenoma (PA) is the most frequent benign salivary gland tumor, but a lip PA is rare. Although this tumor may be definitively diagnosed by imaging or a tissue biopsy if it is reasonably large, PAs on the lip are relatively small, and they present findings that are similar to those of other lip lesions, which can make a preoperative diagnosis difficult. Methods: We analyzed all PAs in the oral region and lesions on the lips treated in our department over the past 20 years, and we discuss them together with the relevant literature. Results: We found that 11.8% (n=6) of the PAs occurred on a lip (upper lip: 9.8%, lower lip: 2.0%), and ~1% of all mass lesions of the lips were PAs. The average size of the lip PAs was 1.5±0.7 cm (range, 0.7-2.2 cm). For preoperative diagnostic assistance, ultrasonography (US) (n=4), magnetic resonance (MR) (n=3), or no imaging (n=2) was used. An excisional biopsy was performed in all cases, and to date, no recurrence or malignant transformation has been observed. Conclusions: Lip PA is relatively rare. Because almost all of these lesions are small, a preoperative diagnosis is more difficult compared to palatal lesions. This tumor is also prone to long-term neglect and has the potential for recurrence and malignant transformation. It is thus necessary to perform an excision that includes the capsule and surrounding tissues, and careful postoperative follow-up should be continued.

Investigation of bone invasion and underlying mechanisms of oral cancer using a cell line-derived xenograft model.

The cancer stroma regulates bone invasion in oral squamous cell carcinoma (OSCC). However, data on normal stroma are limited. In the present study, the effects of gingival and periodontal ligament tissue-derived stromal cells (G-SCs and P-SCs, respectively) and human dermal fibroblasts (HDFs) on bone resorption and osteoclast activation were assessed using hematoxylin and eosin and tartrate-resistant acid phosphatase staining in a cell line-derived xenograft model. The results demonstrated that G-SCs promoted bone invasion and osteoclast activation and inhibited osteoclast proliferation following crosstalk with the human OSCC HSC-3 cell line, whereas P-SCs inhibited bone resorption and promoted osteoclast proliferation in vitro but had a minimal effect on osteoclast activation both in vitro and in vivo following crosstalk with HSC-3 cells. Furthermore, the effects of G-SCs, P-SCs and HDFs on protein expression levels of matrix metalloproteinase (MMP)-9, membrane type 1 MMP (MT1-MMP), Snail, parathyroid hormone-related peptide (PTHrP) and receptor activator of NF-κB ligand (RANKL) in HSC-3 cells in OSCC bone invasion regions were assessed using immunohistochemistry. The results demonstrated that G-SCs had a more prominent effect on the expression of MMP-9, MT1-MMP, Snail, PTHrP, and RANKL, whereas P-SCs only promoted RANKL and PTHrP expression and exerted a minimal effect on MMP-9, MT1-MMP and Snail expression. The potential genes underlying the differential effects of G-SCs and P-SCs on bone invasion in OSCC were evaluated using a microarray, which indicated that cyclin-dependent kinase 1, insulin, aurora kinase A, cyclin B1 and DNA topoisomerase II alpha underlaid these differential effects. Therefore, these results demonstrated that G-SCs promoted bone invasion in OSCC by activating osteoclasts on the bone surface, whereas P-SCs exerted an inhibitory effect. These findings could indicate a potential regulatory mechanism for bone invasion in OSCC.

Deep learning model for analyzing the relationship between mandibular third molar and inferior alveolar nerve in panoramic radiography.

In this study, the accuracy of the positional relationship of the contact between the inferior alveolar canal and mandibular third molar was evaluated using deep learning. In contact analysis, we investigated the diagnostic performance of the presence or absence of contact between the mandibular third molar and inferior alveolar canal. We also evaluated the diagnostic performance of bone continuity diagnosed based on computed tomography as a continuity analysis. A dataset of 1279 images of mandibular third molars from digital radiographs taken at the Department of Oral and Maxillofacial Surgery at a general hospital (2014-2021) was used for the validation. The deep learning models were ResNet50 and ResNet50v2, with stochastic gradient descent and sharpness-aware minimization (SAM) as optimizers. The performance metrics were accuracy, precision, recall, specificity, F1 score, and area under the receiver operating characteristic curve (AUC). The results indicated that ResNet50v2 using SAM performed excellently in the contact and continuity analyses. The accuracy and AUC were 0.860 and 0.890 for the contact analyses and 0.766 and 0.843 for the continuity analyses. In the contact analysis, SAM and the deep learning model performed effectively. However, in the continuity analysis, none of the deep learning models demonstrated significant classification performance.

Reproduction of the Antitumor Effect of Cisplatin and Cetuximab Using a Three-dimensional Spheroid Model in Oral Cancer.

Background/Aim: Cancer research has been conducted using cultured cells as part of drug discovery testing, but conventional two-dimensional culture methods are unable to reflect the complex tumor microenvironment. On the other hand, three-dimensional cultures have recently been attracting attention as in vitro models that more closely resemble the in vivo physiological environment. The purpose of this study was to establish a 3D culture method for oral cancer and to verify its practicality. Materials and Methods: Three-dimensional cultures were performed using several oral cancer cell lines. Western blotting was used for protein expression analysis of the collected cell masses (spheroids), and H-E staining was used for structural observation. The cultures were exposed to cisplatin and cetuximab and the morphological changes of spheroids over time and the expression changes of target proteins were compared. Results: Each cell line formed spheroidal cell aggregates and showed enhancement of cell adhesion molecules over time. H-E staining showed tumor tissue-like structures specific to each cell line. Cisplatin showed concentration-dependent antitumor effects due to loss of cell adhesion and spheroid disruption in each cell line, while cetuximab exhibited antitumor effects that correlated with EGFR expression in each cell line. Conclusion: Spheroids made from oral cancer cell lines appeared to have tumor-like characteristics that may reflect their clinical significance. In the future, it may become possible to produce tumor spheroids from tissue samples of oral cancer patients, and then apply them to drug screening and to develop individualized diagnostic and treatment methods.

Effective deep learning for oral exfoliative cytology classification.

The use of sharpness aware minimization (SAM) as an optimizer that achieves high performance for convolutional neural networks (CNNs) is attracting attention in various fields of deep learning. We used deep learning to perform classification diagnosis in oral exfoliative cytology and to analyze performance, using SAM as an optimization algorithm to improve classification accuracy. The whole image of the oral exfoliation cytology slide was cut into tiles and labeled by an oral pathologist. CNN was VGG16, and stochastic gradient descent (SGD) and SAM were used as optimizers. Each was analyzed with and without a learning rate scheduler in 300 epochs. The performance metrics used were accuracy, precision, recall, specificity, F1 score, AUC, and statistical and effect size. All optimizers performed better with the rate scheduler. In particular, the SAM effect size had high accuracy (11.2) and AUC (11.0). SAM had the best performance of all models with a learning rate scheduler. (AUC = 0.9328) SAM tended to suppress overfitting compared to SGD. In oral exfoliation cytology classification, CNNs using SAM rate scheduler showed the highest classification performance. These results suggest that SAM can play an important role in primary screening of the oral cytological diagnostic environment.

Is attention branch network effective in classifying dental implants from panoramic radiograph images by deep learning?

Attention mechanism, which is a means of determining which part of the forced data is emphasized, has attracted attention in various fields of deep learning in recent years. The purpose of this study was to evaluate the performance of the attention branch network (ABN) for implant classification using convolutional neural networks (CNNs). The data consisted of 10191 dental implant images from 13 implant brands that cropped the site, including dental implants as pretreatment, from digital panoramic radiographs of patients who underwent surgery at Kagawa Prefectural Central Hospital between 2005 and 2021. ResNet 18, 50, and 152 were evaluated as CNN models that were compared with and without the ABN. We used accuracy, precision, recall, specificity, F1 score, and area under the receiver operating characteristics curve as performance metrics. We also performed statistical and effect size evaluations of the 30-time performance metrics of the simple CNNs and the ABN model. ResNet18 with ABN significantly improved the dental implant classification performance for all the performance metrics. Effect sizes were equivalent to "Huge" for all performance metrics. In contrast, the classification performance of ResNet50 and 152 deteriorated by adding the attention mechanism. ResNet18 showed considerably high compatibility with the ABN model in dental implant classification (AUC = 0.9993) despite the small number of parameters. The limitation of this study is that only ResNet was verified as a CNN; further studies are required for other CNN models.

Incidence and Risk of Anti-Resorptive Agent-Related Osteonecrosis of the Jaw after Tooth Extraction: A Retrospective Study.

Bone-modifying agents (BMA) such as bisphosphonates and denosumab are frequently used for the treatment of bone metastases, osteoporosis, and multiple myeloma. BMA may lead to anti-resorptive agent-related osteonecrosis of the jaw (ARONJ). This study aimed to clarify the risk factors for and probabilities of developing ARONJ after tooth extraction in patients undergoing BMA therapy. In this study, the records of 505 target sites of 302 patients undergoing BMA who presented with mandibular fractures at the Department of Oral and Maxillofacial Surgery, Kagawa Prefectural Central Hospital, from March 2014 to January 2022, were retrospectively analyzed for the onset of ARONJ after tooth extraction. The following variables were investigated as attributes: anatomy, health status, and dental treatment. The correlation coefficient was calculated for the success or failure of endodontic surgery for each variable, the odds ratio was calculated for the upper variable, and the factors related to the onset of ARONJ were identified. The incidence rate of ARONJ was found to be 3.2%. Hypoparathyroidism was an important factor associated with ARONJ development. Thus, systemic factors are more strongly related to the onset of ARONJ after tooth extraction than local factors.