Single-cell analysis reveals immune cellular components in odontogenic keratocysts.

OBJECTIVES: Various types of cells comprising a complex and diverse cell population are required for the biological activities of odontogenic keratocyst (OKC). Immune and non-immune cells collaborate via cytokine- or chemokine-mediated communication and direct cell-cell interactions. This study aimed to characterize the immune ecosystem and understand the potential chemotactic role of OKC fibroblasts in immune cell migration. MATERIALS AND METHODS: Mass cytometry of 41 markers was employed for the classification of OKC cells from six OKC samples. Immunofluorescence staining and single-cell RNA sequencing (GSE176351) were used for the detection of fibroblast subpopulations. Enzyme-linked immunosorbent assay and immunofluorescence staining were employed for chemokine detection in hypoxia- and/or HIF-1α inhibitor-treated OKC fibroblasts and tissues. Chemotaxis assay was employed to determine the chemotactic effect of fibroblasts via co-culture with peripheral blood mononuclear cells. A cell communication network was constructed based on the single-cell RNA sequencing data. RESULTS: The characterization of the immune cell types of OKC evidenced the enrichment of macrophages, neutrophils and B cells. The majority (41.5%) of fibroblast subsets consisted of chemokine ligand-enriched myofibroblasts. The activation of the HIF-1α signaling pathway in fibroblasts was associated with chemokine release. The chemokines released by OKC fibroblasts remarkably promoted the migration of peripheral blood mononuclear cells in the co-culture system. Close interactions between myofibroblasts and immune cells were validated by cell-cell interaction analysis. Increased RANKL expression was detected in OKC fibroblasts in the co-culture system with peripheral blood mononuclear cells. CONCLUSIONS: Our results provided deep insights into the immune ecosystem and highlighted the potential chemotactic effects of chemokine-enriched myofibroblasts within OKCs. The close interaction between immune cells and fibroblasts demonstrated in this study may be responsible for the osteoclastogenic effects of OKC fibroblasts.

Salivary non-apoptotic tumoral microvesicles: A potential progressive marker in oral cancer patients.

Tumour cell-secreted microvesicles (MVs) contribute immensely to tumour progression. However, the role of tumoral salivary MVs in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we elucidated the role of non-apoptotic salivary tumoral MVs in OSCC development, especially relating to the migration ability. We purified and compared non-apoptotic salivary tumoral MVs from 63 OSCC patients and orthotopic OSCC mice model. Next, we compared the protein difference between apoptotic and non-apoptotic MVs by Western blot, proteomics and flow cytometry from saliva and CAL27 cells. Finally, we collected the non-apoptotic MVs and co-cultured with normal oral epithelial cells, the migration ability was examined by wound healing assay and Western blot assay. Our results indicated that the levels of non-apoptotic tumoral S-MVs were significantly higher in OSCC patients with T3 to T4 stages than in patients with T1 to T2 stages or healthy donors. In OSCC mice model, we found elevations of non-apoptotic tumoral MVs associated with tumoral volume. EGFR overexpression increased the generation of non-apoptotic tumoral MVs which could significantly promote normal epithelial cell migration. In conclusion, elevated levels of non-apoptotic tumoral S-MVs are associated with clinicopathologic features of OSCC patients, implying that non-apoptotic tumoral S-MVs are a potential progressive marker of OSCC.

Molecularly imprinted polymer-capped wrinkled silica-quantum dot hybrid particles for fluorescent determination of tetra bromo bisphenol A.

A fluorescent probe has been developed for tetra bromo bisphenol A (TBBPA) detection based on molecularly imprinted polymers (MIPs) combined with wrinkled silica nanoparticles (WSNs) and CdTe quantum dot (QD) hybrid particles. The WSNs with large pore sizes were employed as a structural support platform for QD embedding, and MIPs were synthesized on the surface of QD-embedded WSNs. The synthetic procedure was characterized using transmission electron microscopy, Brunauer-Emmett-Teller measurements, X-ray photoelectron spectrometry, Fourier transform infrared spectroscopy, and zeta potential analysis. The MIP-capped wrinkled silica-QD hybrid particles (WSNs-QDs-MIPs) possessed an adsorption capacity of 96.5 mg g-1 with an imprinting factor of 7.9 towards TBBPA. Under the optimum incubation conditions, the fluorescence intensity (λex = 340 nm, λem = 605 nm) was quenched in proportion to added TBBPA in the range 0.025 to 5 µM with a limit of detection of 5.4 nM. The developed probe was successfully applied to the detection of TBBPA in plastic electronic waste samples and the results of this method agreed with those obtained using high-performance liquid chromatography. This method presented a satisfactory selectivity, stability, and reproducibility indicating its potential as a promising probe for TBBPA detection.

Improving the sensitivity and selectivity of sulfonamides electrochemical detection with double-system imprinted polymers.

The extensive use of antibiotics leading to the rapid spread of antibiotic resistance poses high health risks to humans, but to date there is still lack of an on-site detection method of SA residues. In this study, we integrated radical polymerization using sodium p-styrenesulfonate as a functional monomer and the self-polymerization of dopamine to prepare double-system imprinted polymers (DIPs) using sulfonamide antibiotics as templates. We found that the DIPs were semi-interpenetrating polymer networks and introduction of poly(dopamine) improved the selectivity of the imprinted cavities as well as the conductivity. The selectivity and sensitivity of the sensor using DIPs were much higher than those using single-system MIPs. This sensor could determine sulfonamides in complex samples in the presence of structural analogues. The linear range was from 0.01 to 10.00 µmol L-1 with a detection limit of 4.00 nmol L-1. Furthermore, based on the highly selective DIPs and statistics analysis, this method could be used for simultaneous analysis of 4 sulfonamide types in real samples with an accuracy of 94.87 %. This work provides a strategy to improve the selectivity and sensitivity of MIPs based-sensor that can serve as tool for the simultaneous analysis of antibiotic residues in environment samples.

IL-8 Is Upregulated in the Tissue-Derived EVs of Odontogenic Keratocysts.

Background: Interleukin 8 (IL-8) is a chemotactic cytokine released by various cells including leukocytes, endothelial cells, and epithelial cells. IL-8 has multiple functions in inflammation, tumour invasion, or angiogenesis. Human odontogenic cystic lesions are chronic and frequently inflamed. Tissue-derived extracellular vesicles (Ti-EVs) are widely present in various tissues and could more accurately reflect the characteristics of the primary tissue. However, the involvement of IL-8 in Ti-EVs of human odontogenic lesions is still unclear. This study aimed to explore the expression of IL-8 in Ti-EVs of human odontogenic lesions and the potential roles of Ti-EVs that carried IL-8. Methods: Fresh tissue samples of dentigerous cyst (DC, n = 5) and odontogenic keratocyst (OKC, n = 5) were collected for Ti-EVs isolation. Ti-EVs were characterised by transmission electron microscopy and nano-flow cytometry analysis. The cytokine profile of Ti-EVs was explored by cytokine antibody array. The IL-8 expression was examined by immunochemical staining in tissue of odontogenic lesions (DC, n =12; OKC, n =28). Antioxidants (N-acetyl-L-cysteine and diphenyleneiodonium) were employed to treat HaCaT cells, and the expression of IL-8 was detected by enzyme-linked immunosorbent assay. The gene expression of MMP9 was explored by quantitative real-time polymerase chain reaction in co-culture system of fibroblasts of OKC with Ti-EVs. Results: Compared with DC, the expression of IL-8 in Ti-EVs and fixed tissue specimens of OKC was markedly upregulated. The antioxidants decreased the expression level of IL-8 protein in the supernatant of HaCaT cells. The Ti-EVs treatment (10 µg/ml) of fibroblasts significantly induced the MMP9 mRNA expressions in OKC fibroblasts. Conclusions: IL-8 was upregulated in Ti-EVs of OKC and might be involved in the tissue destruction of OKC.

A functional lignin-based nanofiller for flame-retardant blend.

Lignin-based flame retardants represent great promising next-generation flame retardants due to their sustainability, unique aromatic structure, and high charring capability. However, their applications are still limited by the compatibility, processability, and efficiency of flame retardancy. Here, a green functional lignin-based nanofiller (lignin-diethylenetriamine/red phosphorus nanoparticles, Lignin-N-P NPs) was prepared by the chemical modification and co-precipitation. After blending with the commercial acrylonitrile butadiene styrene copolymers (ABS), the physical, chemical, and flame retardant properties of the blends reveal that Lignin-N-P NPs/ABS blend has acceptable processability, mechanical properties, and significantly improved thermal stability and fire performance. Its values of peak heat release rate and total heat released per unit area were significantly dropped 67.8% and 77.5%, respectively. This study will initiate a new design for not only flame retardants but also lignin-based materials.

Single-Cell RNA Sequencing Reveals CXCLs Enriched Fibroblasts Within Odontogenic Keratocysts.

PURPOSE: We aimed to define cell subpopulations of odontogenic keratocyst (OKC), particularly relating to angiogenesis and explored the potential regulation mechanism for angiogenesis. MATERIALS AND METHODS: Single-cell RNA sequencing (scRNA-seq) analysis was investigated on 14,072 cells from 3 donors with OKC. The differential expressed genes, cell trajectory and intercellular communications were evaluated by bioinformatic analysis. Hydrostatic pressure (80 mmHg, 6h) was applied to the primary fibroblasts of OKC and the supernatant was collected for cytokines detection by cytokine antibody array. The chemokine (C-X-C motif) ligand 12 (CXCL12) and CD31 expressions were explored by immunohistochemistry in tissue microarray of OKC. RESULTS: Five different cell types were identified in the epithelium of OKC and 3 different cell types in the OKC fibroblasts were characterized, indicating high intra-lesional heterogeneity. CXCLs were highly enriched in the subset of fibroblasts and showed close interactions with endothelial cells. Hydrostatic pressure (80mmHg) significantly increased CXCL12 secretions in OKC fibroblasts. Stromal CXCL12 expressions were closely related to CD31 expressions of tissue microarray of OKC. CONCLUSION: CXCLs enriched fibroblasts are crucial for angiogenesis of OKCs which could be partially regulated by hydrostatic pressure.

Dual Enzyme-Like Performances of PLGA Grafted Maghemite Nanocrystals and Their Synergistic Chemo/Chemodynamic Treatment for Human Lung Adenocarcinoma A549 Cells.

In recent years, the emergence of non-toxic but catalytically active inorganic nanoparticles has attracted great attention for cancer treatment, but the therapeutic effect has been affected by the limited reactive oxygen species in tumors. Therefore, the combination of chemotherapy and chemodynamic therapy is regarded as a promising therapeutic strategy. In this paper, we reported the preparation and bioactivity evaluation of poly(lactic acid-co-glycolic acid) (PLGA) grafted-γ-Fe2O3 nanoparticles with dual response of endogenous peroxidase and catalase like activities. Our hypothesis is that PLGAgrafted γ-Fe2O3 nanoparticles could be used as a drug delivery system for the anti-tumor drug doxorubicin to inhibit the growth of lung adenocarcinoma A549 cells; meanwhile, based on its mimic enzyme properties, this kind of nanoparticles could be combined with doxorubicin in the treatment of A549 cells. Our experimental results showed that the PLGAgrafted γ-Fe2O3 nanoparticles could simulate the activity of catalase and decompose hydrogen peroxide into H2O and oxygen in neutral tumor microenvironment, thus reducing the oxidative damage caused by hydrogenperoxide to lung adenocarcinoma A549 cells. In acidic microenvironment, PLGA grafted γ-Fe2O3 nanoparticles could simulate the activity of peroxidase and effectively catalyze the decomposition of hydrogen peroxide to generate highly toxic hydroxyl radicals, which could cause the death of A549 cells. Furthermore, the synergistic effect of peroxidase-like activity of PLGA-grafted γ-Fe2O3 nanoparticles and doxorubicin could accelerate the apoptosisand destruction of A549 cells, thus enhancing the antitumor effect of doxorubicin-loaded PLGA-grafted γ-Fe2O3 nanoparticles. Therefore, this study provides an effective nanoplatform based on dual inorganic biomimetic nanozymes for the treatment of lung cancer.

Biodegradable hybrid mesoporous silica nanoparticles for gene/chemo-synergetic therapy of breast cancer.

Mesoporous silica nanoparticles have been extensively explored in anticancer nanomedicine due to their excellent biodegradability, which is one important focus in their further clinical translations. However, the traditional design concepts based on the functional modification with active groups cannot significantly improve the controlled drug release efficiency and anticancer effect. Herein, a molecularly organic-inorganic hybrid mesoporous silica nanoparticle (HMSN) nanocarrier coated with hyaluronic acid (HA) and polyethyleneimine (PEI) was constructed for the gene/chemo-synergetic therapy of breast cancer. Notably, HMSN with tumor-sensitive disulfide bond and targeting ligand HA can be decomposed when it encounters high concentration of glutathione (GSH) and hyaluronidase (HAase). The biodegradability of host molecules and the fast disintegration of the framework in tumor microenvironment can also accelerate the stimuli responsive release of cargos inside the pore space. Furthermore, the grafting of polyethyleneimine (PEI) could increase gene loading efficiency. From the above, the smart approach involves a combination of biodegradability and biological effect and results in synergetic antitumor effect of gene and chemical drug on breast cancer. All these findings demonstrated that HMSN/HA/PEI nanocarriers can be suitable for biomedical application, paving the way to fast development of multi-functional nano-biomedicine.

Fluorescent magnetic PEI-PLGA nanoparticles loaded with paclitaxel for concurrent cell imaging, enhanced apoptosis and autophagy in human brain cancer.

Magnetic nanoparticles are regarded as a promising drug delivery vehicle with the improved efficacy and lowered side effects for antitumor therapy. Herein, the poly lactic-co-glycolic acid (PLGA) modified magnetic nanoplatform was synthesized using superparamagnetic γ-Fe2O3 nanoparticles (MNPs) as a core, and then labelled with polyethylenimine (PEI)-conjugated fluorescein isothiocyanate (FITC), and simultaneously loaded with antitumor drug paclitaxel (PTX) for theranostic analysis of antitumor effects investigated in human brain glioblastoma U251 cells. As a result, the prepared PEI-PLGA-MNPs showed a relatively round sphere with an average size of 80 nm approximately, and the FITC-labeling PEI-PLGA-MNPs were efficiently endocytosed by the U251 cells for cellular imaging. Moreover, the fabricated PEI-PLGA-PTX-MNPs also demonstrated an inhibition of the targeted cell proliferation and migration, and a programmed cell death, via both apoptosis modulating by a burst of reactive oxygen species (ROS) and autophagy with accumulation of autophagosomes and LC3-II signals detected in the treated glioblastoma U251 cells after uptaking. Therefore, the constructed nanoplatform could be effectively applied for simultaneous cellular imaging and drug delivery in human brain glioblastoma treatment in future.

Increased level of cell-derived microparticles in the cyst fluids of odontogenic keratocysts.

The aim of this study was to examine the level and basic characteristics of cell­derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor­κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and tartaric­resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition, in vitro experiments further revealed that CFMPs derived from the OKCs of patients could be taken up by BMMs, leading to a significant increase in the mRNA expression levels of nuclear factor of activated T­cells 1 (NFATc1) and TRAP. Moreover, TRAP­positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony­stimulating factor (M­CSF) and CFMPs with BMMs. On the whole, our findings indicate that patients with OKCs have higher levels of CFMPs compared with patients with DCs and RCs, which may be associated with the bone resorption of OKCs.

The effects of marsupialization on bone regeneration adjacent to keratocystic odontogenic tumors, and the mechanisms involved.

Bone resorption in the jaws is one of the most severe complications of keratocystic odontogenic tumors (KCOTs), and can be treated by either enucleation or marsupialization. However, the effects of marsupialization on bone regeneration adjacent to KCOTs, and the mechanisms involved, are still unclear. In this study, samples of 27 KCOTs were collected (20 before marsupialization and 7 after marsupialization) to detect the expression of bone regeneration-related molecules adjacent to KCOTs by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). The results showed that bone formation was significantly enhanced in the KCOT capsule wall adjacent to bone after marsupialization, as demonstrated by alkaline phosphatase activity assay and immunostaining for bone morphogenetic protein. Moreover, immunohistochemistry revealed that osteoprotegerin (OPG) was up-regulated in the KCOT capsule wall adjacent to bone after marsupialization, while receptor activator of nuclear factor-κB ligand (RANKL) was down-regulated. Real-time qPCR also demonstrated increased expression of OPG after marsupialization, accompanied by a decrease in the expression of osteoclastogenesis-related molecules such as cathepsin K, matrix metalloproteinase-9, NFATc1, RANK and RANKL. This study provides further evidence that marsupialization may promote bone regeneration adjacent to KCOTs partially through regulation of the OPG/RANK/RANKL signaling pathway.

Canonical Wnt signaling acts synergistically on BMP9-induced osteo/odontoblastic differentiation of stem cells of dental apical papilla (SCAPs).

Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. Here, we investigate if BMP9 and Wnt/ß-catenin act synergistically on odontogenic differentiation. Using the immortalized SCAPs (iSCAPs) isolated from mouse apical papilla tissue, we demonstrate that Wnt3A effectively induces early osteogenic marker alkaline phosphatase (ALP) in iSCAPs, which is reduced by ß-catenin knockdown. While Wnt3A and BMP9 enhance each other's ability to induce ALP activity in iSCAPs, silencing ß-catenin significantly diminishes BMP9-induced osteo/odontogenic differentiation. Furthermore, silencing ß-catenin reduces BMP9-induced expression of osteocalcin and osteopontin and in vitro matrix mineralization of iSCAPs. In vivo stem cell implantation assay reveals that while BMP9-transduced iSCAPs induce robust ectopic bone formation, iSCAPs stimulated with both BMP9 and Wnt3A exhibit more mature and highly mineralized trabecular bone formation. However, knockdown of ß-catenin in iSCAPs significantly diminishes BMP9 or BMP9/Wnt3A-induced ectopic bone formation in vivo. Thus, our results strongly suggest that ß-catenin may play an important role in BMP9-induced osteo/ondontogenic signaling and that BMP9 and Wnt3A may act synergistically to induce osteo/odontoblastic differentiation of iSCAPs. It's conceivable that BMP9 and/or Wnt3A may be explored as efficacious biofactors for odontogenic regeneration and tooth engineering.

Increased expression of autophagy-related proteins in keratocystic odontogenic tumours: its possible association with growth potential.

The aim of this study was to evaluate the activation status of autophagy in keratocystic odontogenic tumours (KCOT), and to investigate its possible association with growth potential. We detected the expression of some key autophagy-related proteins in clinical samples of KCOT and radicular cysts and compared then by real-time quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, respectively. The correlation between the autophagy-related proteins tested, and with cell antiapoptotic (Bcl-2) or proliferative (Ki-67) markers in KCOT was explored using Spearman's rank correlation, followed by cluster analysis. The results showed that both the expression of mRNA and the immunoreactivity of the autophagy-related proteins tested were considerably increased in samples of KCOT compared with those in samples of radicular cysts. The correlation analyses showed that the immunostains of autophagy-related proteins in samples of KCOT correlated closely with each other. The immunostains of these autophagy-related proteins also correlated closely with the immunostains of Bcl-2 and Ki-67 in KCOT. More importantly, double-labelling immunofluorescence analyses also showed that the distribution of autophagic and proliferative markers was partially synchronous in samples from KCOT. We have, to our knowledge for the first time, implicated the activation of autophagy in KCOT, and showed its possible association with growth potential.