Down-regulation of microRNA-23b aggravates LPS-induced inflammatory injury in chondrogenic ATDC5 cells by targeting PDCD4

Objective(s): Osteoarthritis (OA), characterized by degradation of articular cartilage, is a leading cause of disability. As the only cell type present in cartilage, chondrocytes play curial roles in the progression of OA. In our study, we aimed to explore the roles of miR-23b in the lipopolysaccharide (LPS)-induced inflammatory injury. Materials and Methods: LPS-induced cell injury of ATDC5 cells was evaluated by the loss of cell viability, enhancement of cell apoptosis, alteration of apoptosis-associated proteins, and release of inflammatory cytokines. Then, miR-23b level after LPS treatment was assessed by qRT-PCR. Next, the effects of aberrantly expressed miR-23b on the LPS-induced inflammatory injury were explored. The possible target genes of miR-23b were virtually screened by informatics and verified by luciferase assay. Subsequently, whether miR-23b functioned through regulating the target gene was validated. The involved signaling pathways were investigated finally.Results: Cell viability was decreased but cell apoptosis, as well as release of inflammatory cytokines, was enhanced by LPS treatment. MiR-23b was down-regulated by LPS and its overexpression alleviated LPS-induced inflammatory injury. PDCD4, negatively regulated by miR-23b expression, was verified as a target gene of miR-23b. Following experiments showed miR-23b alleviated LPS-induced cell injury through down-regulating PDCD4 expression. Phosphorylated levels of key kinases in the NF-κB pathway, as well as expressions of key kinases in the Notch pathways, were increased by PDCD4 overexpression.Conclusion: MiR-23b was down-regulated after LPS treatment, and its overexpression ameliorated LPS-induced inflammatory injury in ATDC5 cells by targeting PDCD4, which could activate the NF-κB/Notch pathways

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

<div><p>The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.</p></div

Physiochemical property of CP and CP/DNA nanoparticles.

<p>(a) SEM images of CP/DNA nanoparticles at CP:DNA weight ratio = 3; (b) the effect of CP:DNA weight ratios on the particle size and the zeta potential of resulting nanoparticles (n = 3; error bars represent standard deviation); (c) size distribution of CP/DNA complexes prepared at the CP:DNA weight ratio = 3 measured by Mastersizer 2000 laser diffractometer; (d) buffering capacities of PEI (25 kDa), CS, and CP copolymers.</p

Images of chondrocytes (a) or synoviocytes (b) transfected with CP/DNA nanoparticles, naked pDNA, CS/DNA nanoparticles, PEI (25 kDa)/DNA nanoparticles, and Lipofectamine™ 2000 as observed under fluorescence microscope or inverted phase contrast microscope.

<p>(40× magnification for upper panel under inverted phase contrast microscope, and 40× magnification for lower panel under fluorescence microscope).</p

Combination effects of graphene and layered double hydroxides on intumescent flame-retardant poly(methyl methacrylate) nanocomposites

A novel intumescent flame-retardant poly(methyl methacrylate) (PMMA) nanocomposite has been prepared via in situ polymerization by incorporating intumescent flame retardants (IFRs), graphene and layered double hydroxides (LDHs). Results from X-ray diffraction (XRD) and transmission electron microscopy (TEM) indicate that a fine dispersion of IFR particles, intercalated LDHs and exfoliated graphene is achieved in the PMMA matrix. Thermal and flammability properties of PMMA nanocomposite were investigated using thermogravimetry, cone calorimetry, limiting oxygen index (LOI) and vertical burning (UL-94). The use of IFRs in combination with graphene and LDHs in the PMMA matrix improves greatly the thermal stability and flame retardant properties of the nanocomposites. The PMMA/IFR/RGO/LDH nanocomposites, filled with 10. wt.% IFRs, 1. wt.% graphene and 5. wt.% LDHs, achieve the LOI value of 28.2% and UL-94 V1 grade. Compared with neat PMMA, the PHRR of PMMA/IFRs/RGO/LDHs is reduced by about 45%, while the mechanical properties of PMMA/IFR/RGO/LDH nanocomposites exhibit almost no deterioration. The results from scanning electronic microscopy (SEM) confirm that the compact and dense intumescent char enhanced with LDHs and graphene nanosheets is formed for the PMMA/IFR/RGO/LDH nanocomposites during combustion, which inhibits the transmission of heat and mass when exposed to flame or heat source, and thus improves the flame retardant properties of the nanocomposites

Porous chitosan scaffolds with embedded hyaluronic acid/chitosan/plasmid-DNA nanoparticles encoding TGF-β1 induce DNA controlled release, transfected chondrocytes, and promoted cell proliferation.

Cartilage defects resulting from traumatic injury or degenerative diseases have very limited spontaneous healing ability. Recent progress in tissue engineering and local therapeutic gene delivery systems has led to promising new strategies for successful regeneration of hyaline cartilage. In the present study, tissue engineering and local therapeutic gene delivery systems are combined with the design of a novel gene-activated matrix (GAM) embedded with hybrid hyaluronic acid(HA)/chitosan(CS)/plasmid-DNA nanoparticles encoding transforming growth factor (TGF)-β1. A chitosan scaffold functioned as the three-dimensional carrier for the nanoparticles. Results demonstrated that scaffold-entrapped plasmid DNA was released in a sustained and steady manner over 120 days, and was effectively protected in the HA/CS/pDNA nanoparticles. Culture results demonstrated that chondrocytes grown in the novel GAM were highly proliferative and capable of filling scaffold micropores with cells and extracellular matrix. Confocal laser scanning microscopy indicated that chondrocytes seeded in the GAM expressed exogenous transgenes labeled with green fluorescent protein. ELISA results demonstrated detectable TGF-β1 expression in the supernatant of GAM cultures, which peaked at the sixth day of culture and afterwards showed a moderate decline. Histological results and biochemical assays confirmed promotion of chondrocyte proliferation. Cell culture indicated no affects on phenotypic expression of ECM molecules, such as GAG. The results of this study indicate the suitability of this novel GAM for enhanced in vitro cartilage tissue engineering

Intracellular distribution of Cy3-labeled pDNA/CP complexes was observed with a confocal fluorescence microscope in chondrocytes (a) and synoviocytes (b).

<p>(Panel 1) 0.5 h post-incubation; (Panel 2) 1 h post-incubation; (Panel 3) 2 h post-incubation; and (Panel 4) 4 h post-incubation. Row A shows the Cy3-labeled pDNA (red); row B shows the lysosomal (green); row C shows the nucleus (blue); and row D shows the overlap of A, B, and C rows content.</p

<i>In vitro</i> transfection efficiency of CP/DNA nanoparticles.

<p>(a) <i>In vitro</i> transfection efficiency of CP/DNA nanoparticles in both chondrocytes and synoviocytes compared to that of naked pDNA, CS/DNA nanoparticles, PEI (25 kDa)/DNA nanoparticles, and Lipofectamine™ 2000 (n = 3; 48 h post-transfection; error bars represent standard deviation). *<i>P</i><0.01 when CP/DNA nanoparticles compared to CS/DNA nanoparticles transfected towards chondrocytes (n = 3); **<i>P</i><0.01 when CP/DNA nanoparticles compared to PEI (25 kDa)/DNA nanoparticles transfected towards synoviocytes (n = 3); # or ## <i>P</i>>0.05 when CP/DNA nanoparticles compared to Lipofectamine™ 2000 transfected towards chondrocytes or synoviocytes (n = 3). (b) Percentage of chondrocytes or synoviocytes transfected <i>in vitro</i> using CP/DNA nanoparticles as measured by flow cytometry 48 h post-transfection. The influence of CP:DNA weight ratios on the transfection efficiency was assessed 48 h post-transfection (n = 3; error bars represent standard deviation).</p

Representative ¹H NMR spectra of chitosan (CS) and CS-<i>g</i>-PEI (CP) in a mixture solution (D₂O/CD₃COOD (V_D2O: V_CD3COOD = 1∶1) at 40°C.

<p>Representative ¹H NMR spectra of chitosan (CS) and CS-<i>g</i>-PEI (CP) in a mixture solution (D₂O/CD₃COOD (V_D2O: V_CD3COOD = 1∶1) at 40°C.</p

Characteristic of prepared CS-g-PEI (CP).

<p>^a calculated from GPC.</p><p>^b calculated from ¹H NMR.</p