Three dimensional graphene promotes the proliferation of cholinergic neurons.

BACKGROUND: An early substantial loss of basal forebrain cholinergic neurons (BFCNs) is a common property of Alzheimer's disease and the degeneration of functional BFCNs is related to learning and memory deficits. As a biocompatible and conductive scaffold for growth of neural stem cells, three-dimensional graphene foam (3D-GF) supports applications in tissue engineering and regenerative medicine. Although its effects on differentiation have been demonstrated, the effect of 3D-GF scaffold on the generation of BFCNs still remains unknown. METHODS: In this study, we used 3D-GF as a culture substrate for neural progenitor cells (NPCs) and demonstrated that this scaffold material promotes the differentiation of BFCNs while maintaining excellent cell viability and proliferation. RESULTS: Immunofluorescence analysis, RT-PCR, western blotting and ELISA revealed that the proportion of BFCNs at 21 days of differentiation reached approximately 30.5% on 3D-GF compared with TCPS group that only presented 9.7%. Furthermore, a cell adhesion study suggested that 3D-GF scaffold enhances the expression of adhesion proteins including vinculin, integrin and N-cadherin. These findings indicate that 3D-GF scaffold materials are preferable candidates for the differentiation of BFCNs from NPCs. CONCLUSIONS: These results suggest new opportunities for the application of 3D-GF scaffold as a neural scaffold for cholinergic neurons therapies based on NPCs.

A precise design strategy for a cell-derived extracellular matrix based on CRISPR/Cas9 for regulating neural stem cell function.

The extracellular matrix (ECM) is a natural microenvironment pivotal for stem cell survival, as well as proliferation, differentiation and metastasis, composed of a variety of biological molecular complexes secreted by resident cells in tissues and organs. Heparan sulfate proteoglycan (HSPG) is a type of ECM protein that contains one or more covalently attached heparan sulfate chains. Heparan sulphate chains have high affinity with growth factors, chemokines and morphogens, acting as cytokine-binding domains of great importance in development and normal physiology. Herein, we constructed endogenous HSPG2 overexpression in mouse embryonic fibroblasts based on the CRISPR/Cas9 synergistic activation mediator system and then fabricated a cell-derived HSPG2 functional ECM (ECMHSPG2). The ECMHSPG2 is capable of enriching basic fibroblast growth factor (bFGF), which binds more strongly than the negative control ECM. With a growing bFGF concentration, ECMHSPG2 could better maintain neural stem cell (NSCs) stemness and promote NSC proliferation and differentiation in culture. These findings provide a precise design strategy for producing a specific cell-derived ECM for biomaterials in research and regenerative medicine.

High-efficiency brain-targeted intranasal delivery of BDNF mediated by engineered exosomes to promote remyelination.

The regeneration of myelin sheaths is the ultimate goal of the treatment of demyelination disease, including multiple sclerosis (MS). However, current drugs for MS mainly target the immune system and can only slow down the disease development and do not promote the differentiation of oligodendrocyte precursor cells (OPCs) abundant in the myelin injury region into mature oligodendrocytes to form a new myelin sheath. Brain-derived neurotrophic factor (BDNF) plays an important role in the regulation of OPC proliferation and differentiation into mature oligodendrocytes. Exosomes, a kind of nanoscale membrane vesicle secreted by cells, can be used as potential therapeutic drug delivery vectors for central nervous system diseases. Here, brain-targeted modification and BDNF intracellular-loaded exosomes were produced through engineering HEK293T cells, which can promote the differentiation of OPCs into mature oligodendrocytes in vitro. The intranasal administration of the brain-targeted engineered exosome-mediated BDNF was a highly effective delivery route to the brain and had a significant therapeutic effect on remyelination and motor coordination ability improvement in demyelination model mice. The combination of intranasal administration with brain-targeted and BDNF-loaded designed exosomes provides a strategy for efficient drug delivery and treatment of central nervous system diseases.

Specific anti-glioma targeted-delivery strategy of engineered small extracellular vesicles dual-functionalised by Angiopep-2 and TAT peptides.

Glioma is one of the primary malignant brain tumours in adults, with a poor prognosis. Pharmacological reagents targeting glioma are limited to achieve the desired therapeutic effect due to the presence of blood-brain barrier (BBB). Effectively crossing the BBB and specifically targeting to the brain tumour are the major challenge for the glioma treatments. Here, we demonstrate that the well-defined small extracellular vesicles (sEVs) with dual-targeting drug delivery and cell-penetrating functions, modified by Angiopep-2 and trans-activator of transcription peptides, enable efficient and specific chemotherapy for glioma. The high efficiency of engineered sEVs in targeting BBB and glioma was assessed in both monolayer culture cells and BBB model in vitro, respectively. The observed high targeting efficiency was re-validated in subcutaneous tumour and orthotopic glioma mice models. After loading the doxorubicin into dual-modified functional sEVs, this specific dual-targeting delivery system could cross the BBB, reach the glioma, and penetrate the tumour. Such a mode of drug delivery significantly improved more than 2-fold survival time of glioma mice with very few side effects. In conclusion, utilization of the dual-modified sEVs represents a unique and efficient strategy for drug delivery, holding great promise for the treatments of central nervous system diseases.

SNHG1/miR-194-5p/MTFR1 Axis Promotes TGFß1-Induced EMT, Migration and Invasion of Tongue Squamous Cell Carcinoma Cells.

Tongue squamous cell carcinoma (TSCC) is a common malignancy with aggressive biological behaviors. Mitochondrial fission regulator 1 (MTFR1), is aberrantly expressed in head and neck squamous cell carcinoma (HNSC), but its role in TSCC remains unclear. We aimed to explore the role of MTFR1 in TSCC. The expression of long non-coding RNA small nucleolar RNA host gene 1 (SNHG1), microRNA-194-5p and MTFR1 in TSCC cells was measured by RT-qPCR. Luciferase reporter assay and RNA pull down assay were applied to confirm the binding capacity between miR-194-5p and SNHG1 (or MTFR1). TSCC cell invasion and migration were accessed by Transwell assays. The protein levels of MTFR1 and epithelial-mesenchymal transition (EMT) markers were examined by western blot. MTFR1 had high expression level in TSCC. MTFR1 knockdown inhibited transforming growth factor ß1 (TGFß1)-induced EMT, migration and invasion of TSCC cells in vitro. MiR-194-5p targeted MTFR1 and negatively regulated its expression. In addition, SNHG1 upregulated the expression of MTFR1 by binding with miR-194-5p. Importantly, SNHG1 promoted EMT, invasion and migration of TSCC cells by upregulating MTFR1. SNHG1/miR-194-5p/MTFR1 axis promotes TGFß1-induced EMT, migration and invasion of cells in TSCC, which could be potential targets for treating TSCC patients.

Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis.

Enhancing neurogenesis of neural stem cells (NSCs) is crucial in stem cell therapy for neurodegenerative diseases. Within the extracellular microenvironment, extracellular matrix (ECM) plays a pivotal role in modulating cell behaviors. However, a single ECM biomaterial is not sufficient to establish an ideal microenvironment. As multifunctional nanocarriers, exosomes display tremendous advantages for the treatments of various diseases. Herein, collagen binding domain peptide-modified exosomes (CBD-Exo) were obtained from the SH-SY5Y cell line infected with lentivirus particles encoding CBD-lysosome associated membrane glycoprotein 2b (CBD-Lamp2b) to improve the binding efficiency of exosomes and ECM. An exosomes-functionalized ECM (CBD-Exo/ECM) was then constructed via the interaction between CBD and collagen in ECM. Then, CBD-Exo/ECM was employed as a carrier for NSCs culture. The results showed that CBD-Exo/ECM can support the neurogenesis of NSCs with the percentage of proliferation marker EdU-positive (35.8% ± 0.47% vs 21.9% ± 2.32%) and neuron maker Tuj-1-positive (55.8% ± 0.47% vs 30.6% ± 2.62%) were both significantly increased in the exosomes-functionalized ECM system. This exosomes-functionalized ECM was capable to promote the cell proliferation and accelerate neuronal differentiation of NSCs, providing a potential biomedical material for stem cell application in tissue engineering and regenerative medicine.

Enhancement of bone formation by BMP-7 transduced MSCs on biomimetic nano-hydroxyapatite/polyamide composite scaffolds in repair of mandibular defects.

This study was to evaluate enhanced bone formation by bone morphogenetic protein-7 (BMP-7) transduced MSCs on nano-hydroxyapatite/polyamide (n-HA/PA) composite scaffolds for bone tissue engineering in repair of mandibular defect. n-HA/PA scaffolds were prepared and rabbit MSCs were separated and expanded; and then infected with adenoviral-mediated BMP-7 in vitro. The MSCs-BMP-7 and MSCs were seeded on the porous scaffolds. Scaffold/MSCs-BMP-7 constructs and scaffold/MSCs constructs were implanted in the defects of rabbits' mandible as the experimental groups A (n = 18) and groups B (n = 18), respectively, the pure scaffolds were implanted as controls (group C, n = 18). Six animals were sacrificed at 4-, 8-, and 16-week postimplantation, respectively. Their mandibles were removed and processed for radiographic, biomechanical tests, histological, and histomorphometric analysis. Group A animals showed greater bone formation and earlier mineralization than group B at 4- and 8-week postimplantation and similarly group B more than group C. However, no difference was found among three groups at 16-week postimplantation. The results of this study suggest that BMP-7 transduced MSCs-n-HA/PA composite could significantly accelerate bone formation in the implant at early stage. BMP-7 mediated ex vivo gene transfer based on MSCs as seed cells, combined with porous n-HA/PA as scaffolds for bone tissue engineering might be an alternative or supplemental approach to repair the mandibular defects.

[Expression of p53 and Ki-67 genes in epithelial dysplasia from old oral mucosa and clinical significance].

OBJECTIVE: To elucidate the relationship between the malignant transformation of oral epithelial dysplasia and the expression of p53 and Ki-67 proteins. METHODS: The expression of p53 and Ki-67 proteins from 60 cases of oral epithelial dysplasia (28 cases of mild epithelial dysplasia, 32 cases of severe epithelial dysplasia) and 30 normal oral mucosa tissue was determined by immunihistochemistry, and a 5-year follow-up study was made. RESULTS: In normal epithelium, Ki-67 expression was only observed in a small number of cells, and no expression of p53 was detected. In the cases of mild epithelial dysplasia, low levels of Ki-67 and p53 expression were detected. In the cases of severe epithelial dysplasia, the expression of p53 and Ki-67 protein was significantly higher than that in the normal oral mucosa and in the mild epithelial cases (P < 0.05). There was a correlation between the malignant transformation and the mutation of p53, Ki-67 in the cases of oral epithelial dysplasia. CONCLUSION: Carcinogenesis of oral mucosa is a continuous and stepwise course; p53 and Ki-67 genes may play an important role in this course.

[Typing human papilloma virus (HPV) infection in the warts of oral mucosa from HIV-positive patients].

OBJECTIVE: To detect and type human papilloma virus (HPV) in the warts of oral mucosa from HIV-positive patients, and better understand the biological characters of these oral warts. METHODS: Polymerase chain reaction (PCR) was used to detect and type HPV infection by consensus HPV primers Gp5+/Gp6+ and specific HPV primers (HPV6/11, 16, 18, 31, 33) in 34 cases of oral mucosa warts from HIV-positive patients. RESULTS: The HPV infection rate was 88.2% by consensus HPV primers Gp5+/Gp6+; the HPV infection rate of HPV6/11, 16, 18, 31 was respectively 47.06%, 11.67%; 2.94%, and 5.88% by specific HPV primers. CONCLUSION: Most lesions of oral warts from HIV-positive patients are associated with the infection of HPV. The low risk HPV6/11 infection is more common than the high risk HPV16, 18, 31.