Immunomodulatory Blood-Derived Hybrid Hydrogels as Multichannel Microenvironment Modulators for Augmented Bone Regeneration.

Autologous blood-derived protein hydrogels have shown great promise in the field of personalized regenerative medicine. However, the inhospitable regenerative microenvironments, especially the unfavorable immune microenvironment, are closely associated with their limited tissue-healing outcomes. Herein, novel immunomodulatory blood-derived hybrid hydrogels (PnP-iPRF) are rationally designed and constructed for enhanced bone regeneration via multichannel regulation of the osteogenic microenvironment. Such double-network hybrid hydrogels are composed of clinically approved injectable platelet-rich fibrin (i-PRF) and polycaprolactone/hydroxyapatite composite nanofibers by using enriched polydopamine (PDA) as the anchor. The polycaprolactone component in PnP-iPRF provides a reinforced structure to stimulate osteoblast differentiation in a proper biomechanical microenvironment. Most importantly, the versatile PDA component in PnP-iPRF can not only offer high adhesion capacity to the growth factors of i-PRF and create a suitable biochemical microenvironment for sustained osteogenesis but also reprogram the osteoimmune microenvironment via the induction of M2 macrophage polarization to promote bone healing. The present study will provide a new paradigm to realize enhanced osteogenic efficacy by multichannel microenvironment regulations and give new insights into engineering high-efficacy i-PRF hydrogels for regenerative medicine.

Comprehensive Analysis of the Role of SLC2A3 on Prognosis and Immune Infiltration in Head and Neck Squamous Cell Carcinoma.

Background: SLC2A3 is upregulated in various cancer types and promotes proliferation, invasion, and metabolism. However, its role in the prognosis and immune regulation of head and neck squamous cell carcinoma (HNSCC) is still obscure. This study is aimed at exploring the prognostic and immunotherapeutic potential of SLC2A3 in HNSCC. Methods: All data were downloaded from TCGA database and integrated via R software. SLC2A3 expression was evaluated using R software, TIMER, CPTAC, and HPA databases. The association between SLC2A3 expression and clinicopathologic characteristics was assessed by R software. The effect of SLC2A3 on survival was analyzed by R software and Kaplan-Meier Plotter. Genomic alterations in SLC2A3 were investigated using the cBioPortal database. Coexpression of SLC2A3 was studied using LinkedOmics and STRING, and enrichment analyses were performed with R software. The relationship between SLC2A3 expression and immune infiltration was determined using TIMER and TISIDB databases. Immune checkpoints and ESTIMATE score were analyzed via the SangerBox database. Results: SLC2A3 expression was upregulated in HNSCC tissues compared to normal tissues. It was significantly related to TNM stage, histological grade, and alcohol history. High SLC2A3 expression was associated with poor prognosis in HNSCC. Coexpression analysis indicated that SLC2A3 mostly participated in the HIF-1 signaling pathway and glycolysis. Furthermore, SLC2A3 expression strongly correlated with tumor-infiltrating lymphocytes in HNSCC. Conclusion: SLC2A3 could serve as a potential prognostic biomarker for tumor immune infiltration in HNSCC.

miR-153-3p inhibited osteogenic differentiation of human DPSCs through CBFß signaling.

Dental pulp stem cells (DPSCs) have multilineage differentiation potential and especially show a great foreground in bone regeneration engineering. The mechanism of osteogenic differentiation of DPSCs needs to be explored exactly. As a kind of endogenous and non-coding small RNAs, microRNAs (miRNAs) play an important role in many biological processes including osteogenic differentiation. However, the mechanism of miR-153-3p in osteogenic differentiation of DPSCs is still unknown. Core-binding factors-beta (CBFß) is a non-DNA-binding factor that combines with the runt-related transcription factor family transcription factors to mediate their DNA-binding affinities, and plays a critical role in regulating osteogenic differentiation. In this study, we explored the mechanisms of miR-153-3p and CBFß in DPSC osteogenesis. The expression of miR-153-3p and CBFß was tested under the osteogenic condition, and the influence led by changing the expression of miR-153-3p or CBFß had also been detected. A luciferase reporter assay confirmed that miR-153-3p directly targeted to CBFß. The osteogenic markers, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and bone morphogenetic protein 2 (BMP2), were tested in protein level or mRNA level. ALP and Alizarin red staining were used to detect the osteoblast activity and mineral deposition. In osteogenic condition, the expressions of CBFß and osteogenic markers were upregulated, whereas that of miR-153-3p was downregulated. miR-153-3p negatively regulated the osteogenic differentiation, and overexpression of CBFß could offset the negative effect of miR-153-3p. Our findings provided a novel strategy for DPSC application in treatment of bone deficiencies and facilitated bone regeneration.

Dose-dependent cytotoxicity induced by pristine graphene oxide nanosheets for potential bone tissue regeneration.

This study was aimed to investigate the toxic effects of pristine graphene oxide (GO) nanosheets on bone-marrow-derived mesenchymal stem cells (BMSCs), a type of traditional seed cells in tissue regeneration engineering. First, a GO suspension was prepared and characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, and Raman shifts. Then, rat BMSCs were isolated and characterized. Subsequently, cell proliferation, membrane integrity, cell cycle, cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were measured. In addition, relevant proteins of the mitochondrial apoptotic pathway and autophagy were analyzed. Our results showed that a high concentration of GO inhibited cell viability and membrane integrity, while cell apoptosis and cell-cycle arrest were induced by GO. Further, GO significantly increased ROS generation and MMP loss with an upregulation of Cleaved Caspase-3, LC3-II/I, and Beclin-1 and a downregulation of Bcl-2 and Caspase3. We concluded that the toxic effects of GO on BMSCs occurred in a dose-dependent manner via the mitochondrial apoptotic pathway and autophagy.

Concentration-dependent cellular behavior and osteogenic differentiation effect induced in bone marrow mesenchymal stem cells treated with magnetic graphene oxide.

The scaffold-free cell sheet plays an important role in stem-cell-based regeneration. Graphene oxide (GO) endows nanoparticles (NPs) with special characteristics and therefore has attracted increasing attention in recent years. However, the existence of toxicity in GO and its derivatives limits their ability to promote osteogenic differentiation. Magnetic graphene oxide (MGO), a novel combination of Fe3 O4 and GO with diverse unique properties, has not been studied in bone tissue engineering. In this study, MGO was fabricated, and the previously undiscovered relationships-including cellular behavior and the effects of osteogenic differentiation and related mechanisms of MGO in rat bone-marrow-derived mesenchymal stem cells (BMSCs)-were investigated for the first time. Here, we found that MGO was not only biocompatible at low concentrations, but also could significantly accelerate osteogenic differentiation in BMSCs. Both the cellular behavior and bone-formation differentiation in BMSCs treated with MGO showed concentration-dependent characteristics. In addition, the regulation of osteogenic differentiation in BMSCs treated with MGO might be involved with the Wnt/ß-catenin and BMP signaling pathways. Furthermore, MGO demonstrated a better ability for osteogenic differentiation in BMSCs than did GO. The current work indicated a significant use for MGO nanocomposite scaffolds in biocompatibility and bone regeneration, which could provide new insight into bone regeneration in the future.

LncRNA GAS5 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition in oral squamous cell carcinoma by regulating the miR-21/PTEN axis.

Growth-arrest-specific transcript 5 (GAS5) functions as a tumor suppressor in a variety of cancers. GAS5 has been reported to be down-regulated in oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the mechanisms of how GAS5 acts as a tumor suppressor in OSCC. qRT-PCR, cell viability, wound-healing, and transwell assays showed that knockdown of GAS5 increased miR-21 expression and promoted proliferation, migration, invasion, and epithelial-mesenchymal transition of OSCC cells. In contrast, overexpression of GAS5 showed the opposite effects. Furthermore, miR-21 overexpression reversed the effect of GAS5. Western blot showed that knockdown of GAS5 suppressed PTEN, while phosphorylation of Akt was promoted. PCNA, cyclinD1, and Ki-67 were up-regulated, indicating enhanced proliferation. E-cadherin was down-regulated, while N-cadherin, vimentin, and snail1 were increased, indicating augmented epithelial-mesenchymal transition. Overexpression of GAS5 regulated these proteins inversely. Overexpression of miR-21 reversed the effect of GAS5 on these proteins. Taken together, GAS5 suppresses proliferation, migration, invasion, and epithelial-mesenchymal transition in OSCC through the miR-21/PTEN axis and might be a novel therapeutic target for OSCC.

MiR-21/STAT3 Signal Is Involved in Odontoblast Differentiation of Human Dental Pulp Stem Cells Mediated by TNF-α.

Dental pulp stem cells (DPSCs), as one type of mesenchymal stem cells (MSCs), have the capability of self-renewal and multipotency to differentiate into several cell lineages, including osteogenesis, odontoblasts, chondrogenesis, neurogenesis, and adipogenesis. It has found that tumor necrosis factor-α (TNF-α) can promote osteogenic differentiation of human DPSCs in our previous studies. Other experimentation revealed that signal transducer and activator of transcription 3 (STAT3) underwent a rapid activation both in osteogenesis and inflammation microenvironment of MSCs in vitro. MicroRNAs (miRNAs or miRs) have been proved in previous studies to regulate MSCs differentiation in vitro. In this study, we identified miR-21 as a key miRNA contributed the functional axis of odontoblast differentiation induced by STAT3. It is observed that the expression of miR-21 and STAT3 increased gradually in low concentration (1-10 ng/mL) of TNF-α, while they were suppressed in high concentration (50-100 ng/mL). The upregulation of miR-21 may facilitate the odontoblast differentiation of DPSCs coordinating with STAT3. SiSTAT3 or treated by the inhibitor of STAT3, cucurbitacin I (Cuc I), significantly increased primary miR-21 expression along with decreased mature miR-21 expression. Meanwhile, the inhibition of miR-21 (anti-miR-21) decreased the activation of STAT3 as well as suppressed the marker proteins of odontoblast differentiation. The results revealed a new function of miR-21, suggesting that miR-21/STAT3 signal may act as a modulator within a complex network of factors to regulate odontoblast differentiation of human DPSCs. It may provide a novel therapeutic strategy to regulate the odontoblast differentiation of DPSCs.

Repeated stimulation by LPS promotes the senescence of DPSCs via TLR4/MyD88-NF-κB-p53/p21 signaling.

Dental pulp stem cells (DPSCs), one type of mesenchymal stem cells, are considered to be a type of tool cells for regenerative medicine and tissue engineering. Our previous studies found that the stimulation with lipopolysaccharide (LPS) might introduce senescence of DPSCs, and this senescence would have a positive correlation with the concentration of LPS. The ß-galactosidase (SA-ß-gal) staining was used to evaluate the senescence of DPSCs and immunofluorescence to show the morphology of DPSCs. Our findings suggested that the activity of SA-ß-gal has increased after repeated stimulation with LPS and the morphology of DPSCs has changed with the stimulation with LPS. We also found that LPS bound to the Toll-like receptor 4 (TLR4)/myeloid differentiation factor (MyD) 88 signaling pathway. Protein and mRNA expression of TLR4, MyD88 were enhanced in DPSCs with LPS stimulation, resulting in the activation of nuclear factor-κB (NF-κB) signaling, which exhibited the expression of p65 improved in the nucleus while the decreasing of IκB-α. Simultaneously, the expression of p53 and p21, the downstream proteins of the NF-κB signaling, has increased. In summary, DPSCs tend to undergo senescence after repeated stimulation in an inflammatory microenvironment. Ultimately, these findings may lead to a new direction for cell-based therapy in oral diseases and other regenerative medicines.

The antimicrobial activity and application of graphene quantum dots in oral medicine / 口腔疾病防治

@#The abuse of antibiotics has been increasing bacterial resistance, which means there is a need to develop methods for the efficient detection and effective treatment of multiresistant bacterial infections. As one of graphene-based materials, graphene quantum dots (GQDs) have distinct mechanical, electrical, and optical properties, including a small size, a large surface area-to-volume ratio, biocompatibility, antimicrobial activity and tunable photoluminescence. Therefore, GQDs are expected to be widely used as antimicrobial materials, drug delivery carriers and photosensitizers in antibacterial applications. In this review, we focus on their synthesis, characteristics and antimicrobial applications in oral medicine.

Expression of miR-21, miR-221, and miR-222 in exosomes of CAL27 tongue squamous cell carcinoma cells / 口腔疾病防治

Objective@# To assess the expression of miR-21, miR-221, and miR-222 in exosomes of CAL27 tongue squamous cell carcinoma cells.@*Methods @# CAL27 tongue squamous cell carcinoma cells and normal human oral keratinocytes (HOKs) were cultured, and then, the cultured supernatant was collected to separate the exosomes. Exosomes were detected by electron microscopy, and the expression levels of miR-21, miR-221, and miR-222 in the exosomes of tongue cancer cells were measured by qRT-PCR. @*Results@#Exosomes existed in the cultured supernatants of CAL27 cells and HOKs. Additionally, the expression levels of miR-21, miR-221, and miR-222 in the exosomes of CAL27 cells were significantly enhanced compared with those in the HOK exosomes (P < 0.05). @*Conclusion @#The expression levels of miR-21, miR-221, and miR-222 were markedly enhanced in the exosomes of CAL27 tongue squamous cell carcinoma cells.

Cellular behaviours of bone marrow-derived mesenchymal stem cells towards pristine graphene oxide nanosheets.

OBJECTIVES: Graphene oxide (GO), the derivative of graphene with unique properties, has attracted much attention for applications in dental implants. The aim of this study was, by two biomimetic cell culture methods, to investigate the quantitative relationship between the concentration of pristine GO nanosheets and their cellular behaviours towards bone marrow-derived mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: The cells were firstly characterized according to their morphology, self-renewal capabilities and multipotency. Subsequently, adhesion density, proliferation, alkaline phosphatase activity and mineralization of BMSCs treated with various concentrations of GO were analysed. In addition, osteogenic-related proteins were measured for further verification of the GO-induced osteogenic differentiation. RESULTS: Pristine GO nanosheets inhibited the proliferation of BMSCs at a high concentration of 10 µg/mL during the first 3 days with two seeding methods and facilitated proliferation of BMSCs at a low concentration of 0.1 µg/mL after 5 days with a sequential-seeding method compared to a co-seeding method. Analogously, osteogenic differentiation was promoted when BMSCs were treated with 0.1 µg/mL of GO. Both the proliferation and differentiation showed concentration-dependent behaviour. Interestingly, Wnt/ß-catenin signalling pathway appeared to be involved in osteogenic differentiation induced by pristine GO nanosheets. CONCLUSIONS: Pristine GO nanosheets at a concentration of 0.1 µg/mL provide benefits to promote BMSCs proliferation and osteogenesis under a sequential-seeding method, contributing to the use of GO for dental implantation.

Downregulation of miR-222 Induces Apoptosis and Cellular Migration in Adenoid Cystic Carcinoma Cells.

Previous studies have shown that miR-222 targets the p53 upregulated modulator of apoptosis (PUMA) to regulate cell biological behavior in some human malignancies. We hypothesized that there was a negative regulation, which might induce apoptosis, between miR-222 and PUMA in adenoid cystic carcinoma (ACC). In this study, the expression levels of miR-222 and the PUMA gene after transfection with antisense miR-222 (As-miR-222) were evaluated by RT-PCR and Western blot assays. Cell proliferation and migratory abilities were detected by CCK-8 and Transwell assays. Cell cycle and apoptosis were analyzed by flow cytometry. Our results showed that, when compared with the control and scramble-transfected groups, the expression of miR-222 in the As-miR-222 group was downregulated, while the expression of PUMA at both mRNA and protein levels was upregulated, cell proliferation and migratory abilities were inhibited, and apoptosis was increased. Our results suggested that As-miR-222 transfection could upregulate the expression of PUMA to induce apoptosis in ACC, providing a new concept for the treatment of ACC.

miR-222 knockdown suppresses epithelial-to-mesenchymal transitionin human oral squamous cell carcinoma.

Tumor metastasis is the main cause of death in patients with oral squamous cell carcinoma (OSCC). Epithelial-to-mesenchymal transition (EMT) is potentially associated with metastasis and histological grading in OSCC. Therefore, the discovery of new strategies to inhibit EMT is potentially valuable for the development of therapies for OSCC. In our previous study, we found that miR-222, which is up-regulated in OSCC, regulates the biological behavior of OSCC cells by targeting the p53-upregulated modulator of apoptosis (PUMA); however, the effect of miR-222 on TGF-ß1-induced EMT in OSCC cells is unclear. In this study, OSCC cell lines CAL-27 and Tca-8113 were incubated with 5 ng/ml of TGF-ß1 to inhibit the expression of E-cadherin, promote the expression of N-cadherin, vimentin, and α-SMA and stimulate achange in cell shape convert from a "cuboidal" epithelial structure into an elongated mesenchymal shape. We found that the expression of miR-222 was up-regulated during TGF-ß1-induced EMT in OSCC cells. In addition, miR-222 knockdown reversed TGF-ß1-induced EMT by targeting PUMA. Our findings indicate that miR-222 plays an important role in OSCC, potentially serving as a novel therapeutic target for the treatment of OSCC.

Research progress of nanomaterials and nanotechnology in cancer radiotherapy sensitization / 口腔疾病防治

@#With the development of nanomaterials and nanotechnology, nanomedicine possesses the vast application prospects in the field of cancer therapy. Although the proportion of radiotherapy in cancer comprehensive therapy is rising, the radiotherapy resistance of cancer cells and the side effects of radiotherapy are the existing problems. Compared with the traditional radiotherapy sensitization, it will present a higher treatment efficiency and lower toxicity to introduce nanomaterials and nanotechnology to cancer radiotherapy. This review elaborates the research of nanomaterials and nanotechnology on cancer radiotherapy sensitization.

Slug inhibition increases radiosensitivity of oral squamous cell carcinoma cells by upregulating PUMA.

As a new strategy, radio-gene therapy was widely used for the treatment of cancer patients in recent few years. Slug was involved in the radioresistance of various cancers and has been found to have an anti-apoptotic effect. This study aims to investigate whether the modulation of Slug expression by siRNA affects oral squamous cell carcinoma sensitivity to X-ray irradiation through upregulating PUMA. Two oral squamous cell carcinoma cell lines (HSC3 and HSC6) were transfected with small interfering RNA (siRNA) targeting Slug and subjected to radiotherapy in vitro. After transfection with Slug siRNA, both HSC3 and HSC6 cells showed relatively lower expression of Slug and higher expression of PUMA. The Slug siRNA transfected cells showed decreased survival and proliferation rates, an increased apoptosis rate and enhanced radiosensitivity to X-ray irradiation. Our results revealed that Slug siRNA transfection in combination with radiation increased the expression of PUMA, which contributed to radiosensitivity of oral squamous cell carcinoma cells. Thus, controlling the expression of Slug might contribute to enhance sensitivity of HSC3 and HSC6 cells toward X-ray irradiation in vitro by upregulating PUMA.