Insights into the role of the N6-methyladenosine reader IGF2BP3 in the progression of oral squamous cell carcinoma and its connection to cell-cycle control.

The genome of oral squamous cell carcinoma (OSCC) has been extensively characterized via bulk sequencing, revealing a multitude of genetic changes. The gene IGF2BP3, which encodes for the insulin-like growth factor 2 mRNA-binding protein 3, has been observed to be highly expressed in several types of cancer. This finding suggests that IGF2BP3 may play a significant role in the initiation and advancement of cancer. Nevertheless, the mechanisms by which IGF2BP3 contribute to OSCC are yet to be fully understood. In this study, we have observed that IGF2BP3 exhibits overexpression in OSCC. Based on our findings from bulk sequencing analysis, we have concluded that IGF2BP3 could potentially serve as a biomarker for predicting poor prognosis in OSCC. Moreover, it has been demonstrated that IGF2BP3 exhibits a significant association with the initiation and advancement of tumors both in vivo and in vitro. The evaluation of IGF2BP3 expression levels in relation to the cell cycle stage was conducted using single-cell RNA sequencing data. Tumor cells characterized by elevated IGF2BP3 expression demonstrated a higher percentage of cells in the G2/M transition phase. This study presents new findings indicating that the molecular target IGF2BP3 can serve as a prognostic indicator for tumors and has an impact on the development and progression of OSCC by influencing the regulation of the cell cycle.

Single-cell RNA sequencing reveals the heterogeneity of epithelial cell and fibroblast cells from non- to metastatic lymph node OTSCC.

Lymph node metastasis (LNM) is one of the common features of oral tongue squamous cell carcinoma (OTSCC). LNM is also taken as a sign of advanced OTSCC and poor survival rate. Recently, single-cell RNA sequencing has been applied in investigating the heterogeneity of tumor microenvironment and discovering the potential biomarkers for helping the diagnosis and prognosticating. Pathogenesis of LNM in OTSCC remains unknown. Specifically, cancer-associated fibroblasts (CAFs) and epithelial tumor cells could foster the progression of tumors. Thus, in this study, we aimed to comprehensively analyze the roles of subpopulations of CAFs and epithelial tumor cells in lymph node metastatic OTSCC using the integration of OTSCC single-cell RNA sequencing datasets. Four distinct subtypes of CAFs, namely vascular CAFs, myofibroblast CAFs, inflammatory CAFs, and growth arrest CAFs were successfully discovered in LNM tumor and confirmed the roles of GAS and PTN pathways in the progression of tumor metastasis. In addition, NKAIN2+ epithelial cells and FN1+ epithelial cells specifically exhibited an upregulation of PTN, NRG, MIF, and SPP1 signaling pathways in the metastatic OTSCC. In doing so, we put forth some potential biomarkers that could be utilized for the purpose of diagnosing and prognosticating OTSCC during its metastatic phase and tried to confirm by immunofluorescence assays.

Metagenomic analysis of healthy and diseased peri-implant microbiome under different periodontal conditions: a cross-sectional study.

BACKGROUND: Peri-implantitis is a polybacterial infection that can lead to the failure of dental implant rehabilitation. This study aimed to profile the microbiome of the peri-implant plaque and estimate the effect of periodontitis on it among 40 Chinese participants with dental implant prostheses and presenting with varying peri-implant and periodontal health states. METHODS: Submucosal plaque samples were collected from four distinct clinical categories based on both their implant and periodontal health status at sampling point. Clinical examinations of dental implant and remaining teeth were carried out. Metagenomic analysis was then performed. RESULTS: The microbiome of the peri-implantitis sites differed from that of healthy implant sites, both taxonomically and functionally. Moreover, the predominant species in peri-implantitis sites were slightly affected by the presence of periodontitis. T. forsythia, P. gingivalis, T. denticola, and P. endodontalis were consistently associated with peri-implantitis and inflammatory clinical parameters regardless of the presence of periodontitis. Prevotella spp. and P. endodontalis showed significant differences in the peri-implantitis cohorts under different periodontal conditions. The most distinguishing function between diseased and healthy implants is related to flagellar assembly, which plays an important role in epithelial cell invasion. CONCLUSIONS: The composition of the peri-implant microbiome varied in the diseased and healthy states of implants and is affected by individual periodontal conditions. Based on their correlations with clinical parameters, certain species are associated with disease and healthy implants. Flagellar assembly may play a vital role in the process of peri-implantitis.

Healing outcomes of open versus closed flap procedures for collagen membrane coverage following immediate dental implant placements with simultaneous guided tissue regeneration.

OBJECTIVE: To compare the outcomes of open healing to complete closure for collagen membrane coverage for immediate implant placements with simultaneous guided bone regeneration (GBR) in two retrospective cohorts. METHODS: The subjects included 118 patients who received Bio-Gide® collagen membrane coverage for immediate implant placements and GBR in 20 anterior and 98 posterior teeth. For 58 patients, gingival flaps were released to achieve full coverage of collagen membrane (CC group). For 60 patients, no efforts were made to release the gingival flaps and collagen membrane was left exposed for open healing (OH group). Antibiotics and analgesics were prescribed for 7 days after surgery. The width of crestal open wounds were measured after surgery (W0), and at 1, 2 and 16 weeks (W16). Changes in bone mass were assessed by cone-beam computed tomography after implant placement and again at W16. Gingival and bone tissues over the implant cover screws were harvested and assessed for 16 patients in the OH group at W16. RESULTS: No wound dehiscence occurred in the CC group from W0 to W16. Both the vertical and horizontal bone dimension changes were not significantly different between the OH and CC group. For the OH group, soft tissue was completely healed at W16 when the initial wound widths were ≤6 mm. For those with initial wound widths ≥ 7 mm, the cover screws were exposed in 5/16 patients at W16 but did not affect the final restorations. Tissue staining showed keratinized mucosa and new bone formation above the dental implant in the OH group. CONCLUSION: Open healing achieved healing outcomes similar to those of complete closure for collagen membrane coverage following immediate implant placements. CLINICAL SIGNIFICANCE: For immediate implant placement requiring bone grafting and collagen membrane coverage, it is unnecessary to release the gingival flaps or use tissue grafts to achieve full coverage of the crestal wounds. Open healing with exposed membrane could achieve similar outcomes with less pain and swelling.

Alveolar Bone Morphologic Predictors for Guided Bone Regeneration Outcome in Anterior Maxilla.

OBJECTIVES: This study aimed to explore the influence of alveolar bone morphologic variables on the outcome of guided bone regeneration (GBR) in the anterior maxilla region. METHODS: Twenty-eight patients who received single maxillary anterior tooth delayed implant placed simultaneously with GBR were recruited. Baseline data including age, gender, implant site, implant brand, and bone graft materials were recorded. The resorption rate of the grafted bone (RRGB), labial bone width at 0 mm, 2 mm, and 4 mm apical to the implant platform at Tn (LBW0Tn, LBW2Tn, LBW4Tn), implant angulation (IA), maximum bone graft thickness (MBGT), bone graft volume (BGV), and the initial bone morphologic variables bone concavity depth (BCD) and bone concavity angulation (BCA) were measured. The Pearson correlation analysis, analysis of variance (ANOVA), and optimal binning method were used to explore the potential predictors for GBR. RESULTS: Among 28 patients, the labial bone width of implant and bone graft volume decreased significantly when measured 6 months after surgery. The mean percentage of RRGB was 49.78%. RRGB was not correlated with gender, age, bone graft material, IA, MBGT, bone graft volume at T1, implant site, and implant brand (P > .05). BCD and BCA were each moderately correlated with RRGB (r = -0.872 [P < .001] and r = 0.686 [P < .001], respectively). A BCD ≥1.03 mm and a BCA <155.30° resulted in a significantly lower percentage of RRGB (P < .001). CONCLUSIONS: A significant grafted bone materials volume reduction was detected after GBR with collagen membrane and deproteinized bovine bone mineral (DBBM). The initial bone morphology can influence GBR outcome, and a bone concavity with a depth ≥1.03 mm and an angulation <155.30° led to a lower RRGB. BCD and BCA can be used as variables to predict the outcome of GBR.

Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors.

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

VE-822 Enhanced Cisplatin Chemotherapy Effects on Head and Neck Squamous Cell Carcinoma Drug-resistant Cells.

PURPOSE: The study aimed to assess the effect of p-ATR inhibitor VE-822 in the combination chemotherapy with cisplatin of head and neck squamous cell carcinoma and to explore the possible mechanism. METHODS: The DNA damage levels were determined by comet assay and western blot experiments in cisplatin-resistant and sensitive cell lines. The IC50 value changes after combination treatment with VE-822 in cisplatin sensitive and resistant cell lines were detected by the CCK-8 test. The effects of VE-822 combined with cisplatin on proliferation ability, colony formation ability, migration ability, cell apoptosis and cell cycle changes were observed in vitro. In vivo, the combination treatment effect was verified in the subcutaneous xenograft models of nude mice. Besides, the mechanism of VE-822 assisting cisplatin in chemotherapy was explored by comet assay, western blotting and immunohistochemical experiments. RESULTS: The increased expression of the p-ATR protein was related to the DNA damage repair pathway in head and neck squamous cell carcinoma cisplatin-resistant cells. VE-822 inhibited cell proliferation, colony formation and migration abilities and improved the cisplatin chemotherapeutic effects in subcutaneous xenograft models of nude mice by inhibiting the p-ATR expression and blocking DNA damage repair pathway. CONCLUSIONS: The p-ATR expression increased in head and neck squamous cell carcinoma cisplatinresistant cells. VE-822 significantly enhanced the therapeutic effect in cisplatin resistant head and neck squamous cell carcinoma by inhibiting p-ATR expression in vivo and in vitro.

Spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells maintain pluripotency of stem cells by regulating hypoxia-inducible factors

BACKGROUND: Spontaneous spheroid culture is a novel three-dimensional (3D) culture strategy for the rapid and efficient selection of progenitor cells. The objectives of this study are to investigate the pluripotency and differentiation capability of spontaneous spheroids from alveolar bone-derived mesenchymal stromal cells (AB-MSCs); compare the advantages of spontaneous spheroids to those of mechanical spheroids; and explore the mechanisms of stemness enhancement during spheroid formation from two-dimensional (2D) cultured cells. METHODS: AB-MSCs were isolated from the alveolar bones of C57BL/6 J mice. Spontaneous spheroids formed in low-adherence specific culture plates. The stemness, proliferation, and multi-differentiation capacities of spheroids and monolayer cultures were investigated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, alkaline phosphatase (ALP) activity, and oil-red O staining. The pluripotency difference between the spontaneous and mechanical spheroids was analyzed using RT-qPCR. Hypoxia-inducible factor (HIFs) inhibition experiments were performed to explore the mechanisms of stemness maintenance in AB-MSC spheroids. RESULTS: AB-MSCs successfully formed spontaneous spheroids after 24 h. AB-MSC spheroids were positive for MSC markers and pluripotency markers (Oct4, KLF4, Sox2, and cMyc). Spheroids showed higher Ki67 expression and lower Caspase3 expression at 24 h. Under the corresponding conditions, the spheroids were successfully differentiated into osteogenic and adipogenic lineages. AB-MSC spheroids can induce neural-like cells after neurogenic differentiation. Higher expression of osteogenic markers, adipogenic markers, and neurogenic markers (NF-M, NeuN, and GFAP) was found in spheroids than in the monolayer. Spontaneous spheroids exhibited higher stemness than mechanical spheroids did. HIF-1α and HIF-2α were remarkably upregulated in spheroids. After HIF-1/2α-specific inhibition, spheroid formation was significantly reduced. Moreover, the expression of the pluripotency genes was suppressed. CONCLUSIONS: Spontaneous spheroids from AB-MSCs enhance stemness and pluripotency. HIF-1/2α plays an important role in the stemness regulation of spheroids. AB-MSC spheroids exhibit excellent multi-differentiation capability, which may be a potent therapy for craniomaxillofacial tissue regeneration.

DDX59-AS1 is a prognostic biomarker and correlated with immune infiltrates in OSCC.

Background: lncRNAs play a critical role in multiple steps of gene regulation associated with tumor progression. However, the engagement of DDX59-AS1, a lncRNA, remains equivocal, particularly in oral squamous cell carcinoma (OSCC). In this study, the expression of DDX59-AS1 and its association with immune infiltration were investigated, and its prognostic value in OSSC was evaluated. Methods: OSCC patients were collected from The Cancer Genome Atlas (TCGA) database. The expression of DDX59-AS1 in OSCC and healthy tissue was compared using Wilcoxon rank sum test. The relationship between DDX59-AS1 and clinicopathological features was analyzed using Logistic regression. Gene ontology (GO) terminology analysis, gene set enrichment analysis (GSEA), and single sample GSEA (ssGSEA) were utilized to interpret the enrichment pathway and functionality and to quantify the immune cell infiltration of DDX59-AS1. The correlation between survival and DDA59-AS1 was evaluated by Kaplan-Meier analysis and Cox regression. The prognostic impact of DDX59-AS1 was predicted by the nomogram based on Cox multivariate analysis. Results: High expression of DDX59-AS1 was significantly correlated with T stage, clinical stage, race, and age (p < 0.05). Multivariate survival analysis demonstrated that the high expression of DDX59-AS1 was associated with lower overall and specific survival rates. The prognosis prediction was validated by the nomogram and calibration curves. The expression of DDX59-AS1 was negatively correlated with Mast cells, Tfh, T cells, Treg, and B cells, and positively related with the Tgd infiltration level. Conclusion: DDX59-AS1 played a crucial role in the progression and prognosis of OSCC and was potentially a predictive biomarker for OSCC.

Dual Biosignal-Functional Injectable Microspheres for Remodeling Osteogenic Microenvironment.

Bone defect regeneration depends on the population and lifespan of M2 macrophages, which are regulated by dual signals generated by the "physical" spatial configuration of biological tissues and "molecular" chemokines. Herein, inspired by the reprogramming of macrophages, immunoengineered porous microspheres are constructed to accelerate bone repair through the regulation of both "physical" and "molecular" signals. The porous structure of injectable poly (l-lactic acid) (PLLA) microspheres prepared by the microfluidic technique provides a "physical signal" for osteogenic differentiation. Additionally, interleukin (IL)-4-loaded liposomes (Ls) are modified on PLLA microspheres through amide bonds to produce IL-4/Ls/PLLA microspheres, providing a "molecular signal" in stimulating the differentiation of macrophages to M2 type. It is confirmed that IL-4/Ls/PLLA microspheres could induce M2-macrophages polarization and potentiate osteoblast proliferation and differentiation while coculturing with macrophages and osteoblasts in vitro. Besides, IL-4/Ls/PLLA microspheres are proved to promote bone defect regeneration by inducing the conversion of M1 macrophages to M2 through dual biosignal-functional regulation in both the calvaria defect and maxillary sinus defect models. Overall, the immuno-reprogrammed IL-4/Ls/PLLA microspheres achieve the precise immuno-reprogramming of macrophages by dual biosignal-functional regulation. This immune reengineering strategy paves a way for clinical bone defect treatment.

Nomogram to predict the prognosis of parotid gland mucoepidermoid carcinoma: a population-based study of 1306 cases.

BACKGROUND: Mucoepidermoid carcinoma (MEC) is a common cancer in the oral salivary gland malignancy, which mainly occurs in the parotid gland. The aim of this study is to identify independent prognostic factors and establish a nomogram model for parotid gland mucoepidermoid carcinoma (P-MEC) patients using the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database. METHOD: Patients with P-MEC were selected from between 2004 and 2015. The overall survival (OS) and cancer-specific survival (CSS) rates were estimated using the Kaplan-Meier method with the log-rank test. Univariate and multivariate Cox proportional hazards regression analyses were performed to identify the independent prognostic factors. RESULTS: A total of 1,306 patients with P-MEC were enrolled. Age, grade, T stage, N stage, M stage, chemotherapy, and surgery type were independent prognostic factors for OS and CSS. A nomogram for OS was formulated based on these independent prognostic factors and validated using an internal bootstrap resampling approach, which showed that the nomogram exhibited a sufficient level of discrimination according to the C-index (0.877, 95% CI [0.855-0.898]). CONCLUSION: Several prognostic factors for P-MEC were identified. The nomogram developed in this study accurately predicted the 5- and 10-year OS rates of American patients with P-MEC based on individual characteristics. Risk stratification using the survival nomogram can optimize individual therapies and follow-up.

Effect of GARP on osteogenic differentiation of bone marrow mesenchymal stem cells via the regulation of TGFß1 in vitro.

Mesenchymal stem cells (MSCs), which have multipotential differentiation and self-renewal potential, are possible cells for tissue engineering. Transforming growth factor ß1 (TGFß1) can be produced by MSCs in an inactive form, and the activation of TGFß1 functions as an important regulator of osteogenic differentiation in MSCs. Recently, studies showed that Glycoprotein A repetitions predominant (GARP) participated in the activation of latent TGFß1, but the interaction between GARP and TGFß1 is still undefined. In our study, we successfully isolated the MSCs from bone marrow of rats, and showed that GARP was detected in bone mesenchymal stem cells (BMSCs). During the osteogenic differentiation of BMSCs, GARP expression was increased over time. To elucidate the interaction between GARP and TGFß1, we downregulated GARP expression in BMSCs to examine the level of active TGFß1. We then verified that the downregulation of GARP decreased the secretion of active TGFß1. Furthermore, osteogenic differentiation experiments, alkaline phosphatase (ALP) activity analyses and Alizarin Red S staining experiments were performed to evaluate the osteogenic capacity. After the downregulation of GARP, ALP activity and Alizarin Red S staining significantly declined and the osteogenic indicators, ALP, Runx2, and OPN, also decreased, both at the mRNA and protein levels. These results demonstrated that downregulated GARP expression resulted in the reduction of TGFß1 and the attenuation of osteoblast differentiation of BMSCs in vitro.

Implants placed simultaneously with maxillary sinus floor augmentation in the presence of antral pseudocysts: Presentation of a case series.

OBJECTIVE: To report the outcome of simultaneous placement of dental implants with maxillary sinus elevation in the presence of antral pseudocysts. METHOD AND MATERIALS: This case series involved 14 patients with 28 implants placed simultaneously with maxillary sinus elevation. The psuedocysts were treated by cystic fluid extraction. Postoperative examinations were performed at 3, 6, and 12 months. RESULTS: All restorations were finished 6 to 9 months after implant placement. Nine antral pseudocysts disappeared and five decreased in size. All implants were deemed successful at 1 year postoperatively, showing osseointegration and masticatory function. No mobility was found during the follow-up period. CONCLUSION: In the absence of maxillary sinus infection, the combination of cystic fluid extraction, maxillary sinus elevation, and immediate implantation showed an acceptable clinical outcome in this series of patients.

Laser beam melting 3D printing of Ti6Al4V based porous structured dental implants: fabrication, biocompatibility analysis and photoelastic study.

Fabricating Ti alloy based dental implants with defined porous scaffold structure is a promising strategy for improving the osteoinduction of implants. In this study, we use Laser Beam Melting (LBM) 3D printing technique to fabricate porous Ti6Al4V dental implant prototypes with three controlled pore sizes (200, 350 and 500 µm). The mechanical stress distribution in the surrounding bone tissue is characterized by photoelastography and associated finite element simulation. For in-vitro studies, experiments on implants' biocompatibility and osteogenic capability are conducted to evaluate the cellular response correlated to the porous structure. As the preliminary results, porous structured implants show a lower stress-shielding to the surrounding bone at the implant neck and a more densed distribution at the bottom site compared to the reference implant. From the cell proliferation tests and the immunofluorescence images, 350 and 500 µm pore sized implants demonstrate a better biocompatibility in terms of cell growth, migration and adhesion. Osteogenic genes expression of the 350 µm group is significantly increased alone with the ALP activity test. All these suggest that a pore size of 350 µm provides an optimal provides an optimal potential for improving the mechanical shielding to the surrounding bones and osteoinduction of the implant itself.