Leyu Wang

Cell-compatible hydrogels based on a multifunctional crosslinker with tunable stiffness for tissue engineering

This paper reports a functionalized cationic poly(amidoamine) crosslinker synthesized by Michael addition of peptide mimetic agmatine to N,N′-bis(acryloyl)cystamine. A polyethylene glycol diacrylate (PEGDA) hybrid hydrogel was developed using a functionalized crosslinker with cell adhesion capability and tunable stiffness. This engineered hydrogel presents a 3-D in-growth of cells and the cryosection shows remarkable morphology of the gel. The tunable stiffness of this hydrogel can regulate the osteogenic differentiation of mesenchymal stem cells from RT-PCR. Both in vitro and in vivo tests demonstrate adjustable degradation rate and biocompatibility. The crosslinker and its hydrogels hold promising potential for tissue engineering applications.

Low concentration of lipopolysaccharide acts on MC3T3‐E1 osteoblasts and induces proliferation via the COX‐2‐independent NFκB pathway

The translocations of lipopolysaccharide (LPS) from the gut and its effects on bone healing are usually of clinical interest during bone fracture. As already widely stuided, Cyclooxygenase‐2 (COX‐2) is a key enzyme for prostaglandin E2 (PGE2) production, which induces the nuclear factor kappa B (NFκB) activation and is beneficial to fracture healing. In order to know their roles in skeletal regeneration, mouse MC3T3‐E1 osteoblasts were treated with NFκB inhibitor BAY 11‐7082 and sc791 (a selective COX‐2 inhibitor), in the presence of LPS. Interestingly, LPS could induce osteoblasts proliferation through increasing NFκB activation and translocation. This induction was not related to COX‐2 expression, suggesting that LPS‐induced NFκB activiation is independent of COX‐2. It is possible that low concentration of LPS can act as a stimulating factor of the NFκB pathway in nonstimulated cells such as osteoblasts. COX‐2 is not necessary for the NFκB pathway during LPS‐induced proliferation of osteoblasts since sc791 had no effects on this induction. These studies provide insight into a potential mechanism by which LPS can affect bone tissue repair in the initial phase of inflammation. Copyright

Mitochondrial AIF protein involved in skeletal muscle regeneration

The mitochondrial flavoprotein apoptosis‐inducing factor (AIF) has proved to be either the main mediator of apoptosis or an anti‐apoptotic factor via its putative oxidoreductase and peroxide scavenging activities. We report here that 100 µM hydrogen peroxide (H2O2) induced the proliferation of C2C12 myoblasts and over‐expression of AIF simultaneously in vitro. Immunofluorescence showed that the over‐expression of AIF was located in the cytoplasm. The immunopositive AIF was detected in nuclei 27 days after denervation of skeletal muscle, but in the cytoplasm it was detected 27 days after fiber‐damaged skeletal muscle. AIF may be a factor involved in skeletal muscle regeneration. Copyright

Mechanical stretch regulates microRNA expression profile via NF-κB activation in C2C12 myoblasts

MicroRNAs (miRNAs/miRs) and nuclear factor (NF)-κB activation are involved in mechanical stretch-induced skeletal muscle regeneration. However, there are a small number of miRNAs that have been reported to be associated with NF-κB activation during mechanical stretch-induced myogenesis. In the present study, C2C12 myoblasts underwent cyclic mechanical stretch in vitro, to explore the relationship between miRNA expression and NF-κB activation during stretch-mediated myoblast proliferation. The results revealed that 10% deformation, 0.125 Hz cyclic mechanical stretch could promote myoblast proliferation. The miRNA expression profile was subsequently altered; miR-500, −1934, −31, −378, −331 and −5097 were downregulated, whereas miR-1941 was upregulated. These miRNAs were all involved in stretch-mediated myoblast proliferation. Notably, the expression of these miRNAs was reversed following treatment of 0.125 Hz mechanically stretched C2C12 cells with NF-κB inhibitors, which was accompanied by C2C12 cell growth suppression. Therefore, the present study is the first, to the best of our knowledge, to demonstrate that the NF-κB-dependent miRNA profile is associated with mechanical stretch-induced myoblast proliferation.

Nix Protein Positively Regulates NF-κB Activation in Gliomas

Previous reports indicate that the NIX/BNIP3L gene acts as a pro-apoptotic factor by interacting with BCL2 and BCL-XL, playing an important role in hypoxia-dependent cell death and acting as a tumor suppressor. However, many studies also showed that NIX is linked to a protective role and cell survival in cancer cells. Nuclear factor-κB (NF-κB) can attenuate apoptosis in human cancers in response to chemotherapeutic agents and ionizing radiation. We observed an absence of i-κBα (NF-κB activation inhibitor) expression, but a greater expression of Nix and p-NF-κB proteins in the Nix-wt U251 cells, which was not observed in the Nix-kn cells under hypoxic conditions. Using electrophoretic mobility shift assay (EMSA) and luciferase detection, the activation of NF-κB was detected only in the Nix-wt U251 cells with hypoxia. These data imply that Nix protein might play a role in the positive regulation of the NF-κB pathway. Moreover, 46 cases of glioma also showed high levels of Nix protein expression, which was always accompanied by high p-NF-κB expression. Patients with Nix (+) showed less tissue apoptosis behavior in glioblastoma (GBM), unlike that observed in the Nix-negative patients (−). The same apoptotic tendency was also identified in anaplastic astrocytoma (AA) groups, but not in astrocytoma (AS). On analyzing the Kaplan-Meier curve, better tumor-free survival was observed only in cases of astrocytoma, and not in AA and GBM. Thus, our study indicates that Nix protein might have multiple functions in regulating glioma behaviors. In the low-grade gliomas (astrocytoma) with low expression of NF-κB, the cell death-inducing function that occurs through a Bax mechanism might predominate and act as a tumor suppressor. While in the malignant gliomas (AA and GBM), with higher expression of the NIX gene and with activity of the NF-κB pathway, the oncogene function of Nix was predominant.

Long-term intraperitoneal injection of lipopolysaccharide induces high expression of Id2 in the brain of mice

Lipopolysaccharide (LPS) from gram negative bacteria plays an important role in the pathophysiology of neurodegenerative diseases. Many evidences showed that LPS-induced neuroinflammation is related to upregulation of NF-kappaB. Here, we report that long-term treatment of lower dosage LPS mainly causes upregulation of Id2 protein. As an inhibitor of cell differentiation, Id2 plays an import role in adult olfactory neurogenesis. However, Id2 protein in brain acts as two edges in a sword, persist over-expression of Id2 in brain can induce neurodamages and may be related to neurodegeneration.

Thermally sensitive conductive hydrogel using amphiphilic crosslinker self-assembled carbon nanotube to enhance neurite outgrowth and promote spinal cord regeneration

Spinal cord injury leads to severe sensory or motor damage in the human body. Efforts have been made to activate the nerve function by trying physical and biochemical strategies. Carbon nanotubes as conductive materials have been used to transmit electrical signals to improve cell–cell communication and cross-talk, besides providing an extracellular scaffold for neurons. We reported a thermally sensitive hydrogel using copolymerization of n-isopropylacrylamide, the oligomeric amphiphilic crosslinker of polyethylene glycol diacrylate–dodecylamine–1-(2-aminoethyl)piperazine (PEGDA–DD–AEP), and single-walled carbon nanotubes. We hypothesized that carbon nanotubes in the gel could improve neurite outgrowth and nerve regeneration. In order to overcome the aggregation issue of carbon nanotubes, the hydrophobic chains of the amphiphilic crosslinker were used to stabilize the nanotubes. The carbon nanotube–poly(n-isopropylacrylamide) (PNIPAAM) hydrogel was injectable and improved the electrical conductivity. We found that the hydrogel may have potential to promote the growth of SH-SY5Y cells, with significant neurite outgrowth while electrical stimulation was given. In a spinal cord injury model, creating a 1 mm × 1 mm × 1 mm cavity at C7, we found that the hydrogel promoted nerve tissue regeneration and reduced the formation of scar tissue. Therefore, the hydrogel may be a potential repairing biomaterial for neuron network reconstruction and spinal cord regeneration.

Mussel-inspired dual-functional PEG hydrogel inducing mineralization and inhibiting infection in maxillary bone reconstruction

Infection compromises healing process after bone fracture. An anti-bacterial bone graft synthesized from polymer and mineralization components is becoming preferable for its accessibility and low cost and tunable chem-physic properties. In this study, mussel-inspired polydopamine (PDA) was used to synthesize in-situ silver nanoparticles (AgNPs) and mineralization on polyethylene hydrogel (PEG). With dual functions of anti-bacteria and graft mineralization, we found the hydrogel (AgNPs/PDA) promoted bone generation and show significant antibacterial activity. Specifically, the gel upregulated the expression of osteogenic genes of bone sialoprotein gene, alkaline phosphatase, osteocalcin and runt-related transcription factor 2. It also significantly inhibited the growth of Staphylococcus aureus and Escherichia coli. In vivo the AgNPs/PDA gel could repair maxillary bone defect efficiently.

A simple 3D cryogel co-culture system used to study the role of CAFs in EMT of MDA-MB-231 cells

Breast cancer is one of the most common epithelial tumors and can seriously endanger womens health. Studies have shown that carcinoma-associated fibroblasts (CAFs) possess myofibroblast features and play an important role in the growth and metastasis of breast cancer. Because of the lack of an applicable animal model, the exact role of CAFs in tumor metastasis is still unclear. In this research, a simple three-dimensional (3D) cryogel co-culture system was developed to evaluate the role of CAFs in breast cancer metastasis. It was found that CAFs could promote epithelial–mesenchymal transition of MDA-MB-231 cells (human breast cancer cells) in a 3D cryogel co-culture system in vitro. Then the co-culture human breast cancer system was used to construct a xenograft tumor mouse model in vivo. It was found that CAFs could promote deterioration and metastasis of MDA-MB-231 cells in a xenograft tumor mouse model in vivo.

Delivery Efficiency of miR-21i-CPP-SWCNT and Its Inhibitory Effect on Fibrosis of the Renal Mesangial Cells

MicroRNA 21 miR-21 was proved to cause renal fibrosis and the inhibition of miR-21 would improve the poor prognosis in renal cell carcinoma diseases. The complementary oligonucleotide of mature miR-21 was considered to be an effective intracellular miR-21 inhibitor miR-21i. The directly effective delivery of miR-21i into fibrotic cell is a facile method for treatment of renal fibrosis. Herein, the miR-21i-CPP-SWCNT delivery system, synthesized via single-walled carbon nanotube SWCNT and cell-penetrating peptide CPP, was taken as a novel fibrosis-targeting therapeutic carrier. The miR-21i and CPP firstly bind together via electrostatic forces, and subsequently miR-21i-CPP binds to the surface of SWCNTs via hydrophobic forces. CPP could endow the delivery system with targeting property, while SWCNT would enhance its penetrating ability. The exogenous miR-21i released from the designed miR-21i-CPP-SWCNTs had successfully inhibited the expression of fibrosis-related proteins in renal mesangial cells RMCs. We found that the expression of TGF-β1 proteins was more sensitive to miR-21i-CPP-SWCNT than the expression of α-SMA proteins.