Poly(lactide-<i>co</i>-glycolide)/Fibrin Gel Construct as a 3D Model to Evaluate Gene Therapy of Cartilage in Vivo

Combination of gene therapy with tissue engineering can enhance the interplay between cells and matrix, leading to better restoration and regeneration of tissues and organs in vivo. In this study the PLGA/fibrin gel hybrids were employed to load lipofectamine/pDNA-TGF-β1 complexes and mesenchymal stem cells (MSCs) (experimental group), acting as a cartilage-mimetic tissue platform. The gene complexes distributed more evenly in the hybrid scaffolds, whereas they adhered onto the pore walls of the PLGA sponges. The filled fibrin gel rendered gene release in a slower manner, too. Moreover, the fibrin gel entrapped MSCs and contributed to a higher cell loading density in the hybrid constructs. In vivo assay showed that in the defects implanted with the experimental constructs both gene and protein expression levels of TGF-β1 were significantly higher than those of the fibrin-free group at weeks 1, 3, and 6 after surgery. The full articular cartilage defects repaired by the experimental group for 12 w were resurfaced by neo-tissues with a similar thickness, cell arrangement, and color to the normal neighboring cartilage and abundant glycosaminoglycans

Molecular Mechanisms of Reduced Nerve Toxicity by Titanium Dioxide Nanoparticles in the Phoxim-Exposed Brain of <i>Bombyx mori</i>

<div><p><i>Bombyx mori</i> (<i>B. mori</i>), silkworm, is one of the most important economic insects in the world, while phoxim, an organophosphorus (OP) pesticide, impact its economic benefits seriously. Phoxim exposure can damage the brain, fatbody, midgut and haemolymph of <i>B. mori</i>. However the metabolism of proteins and carbohydrates in phoxim-exposed <i>B. mori</i> can be improved by Titanium dioxide nanoparticles (TiO₂ NPs). In this study, we explored whether TiO₂ NPs treatment can reduce the phoxim-induced brain damage of the 5th larval instar of <i>B. mori</i>. We observed that TiO₂ NPs pretreatments significantly reduced the mortality of phoxim-exposed larva and relieved severe brain damage and oxidative stress under phoxim exposure in the brain. The treatments also relieved the phoxim-induced increases in the contents of acetylcholine (Ach), glutamate (Glu) and nitric oxide (NO) and the phoxim-induced decreases in the contents of norepinephrine (NE), Dopamine (DA), and 5-hydroxytryptamine (5-HT), and reduced the inhibition of acetylcholinesterase (AChE), Na⁺/K⁺-ATPase, Ca²⁺-ATPase, and Ca²⁺/Mg²⁺-ATPase activities and the activation of total nitric oxide synthase (TNOS) in the brain. Furthermore, digital gene expression profile (DGE) analysis and real time quantitative PCR (qRT-PCR) assay revealed that TiO₂ NPs pretreatment inhibited the up-regulated expression of <i>ace1, cytochrome c</i>, <i>caspase-9</i>, <i>caspase-3</i>, <i>Bm109</i> and down-regulated expression of <i>BmIap</i> caused by phoxim; these genes are involved in nerve conduction, oxidative stress and apoptosis. TiO₂ NPs pretreatment also inhibited the down-regulated expression of <i>H⁺ transporting ATP synthase</i> and <i>vacuolar ATP synthase</i> under phoxim exposure, which are involved in ion transport and energy metabolism. These results indicate that TiO₂ NPs pretreatment reduced the phoxim-induced nerve toxicity in the brain of <i>B. mori</i>.</p></div

Effects of TiO₂ NPs on nerve conduction in the brain of phoxim-exposed fifth-instar larvae.

<p>*<i>p</i><0.05, **<i>p</i><0.01, and ***<i>p</i><0.001. Values represent means ± SEM (<i>N</i> = 5). (a) Neurotransmitter contents, (b) Enzyme activity.</p

Comparison between fold-difference with qRT-PCR results and DGE assay in each group.

<p>*p<0.05, **p<0.01, and ***p<0.001.</p><p>Values represent means ± SEM (<i>n</i> = 5).</p

Histopathology of the brain tissue in fifth-instar larvae after phoxim exposure 48 h.

<p>(a) Control; (b) TiO₂ NPs; (c) Phoxim; (d) TiO₂ NPs + Phoxim. Green arrows indicate breakage of nerve fibers, yellow arrows show adipose degeneration, blue arrows indicate cell debris.</p

Effects of TiO₂ NPs on oxidative stress in brain of phoxim-exposed fifth-instar larvae.

<p>*<i>p</i><0.05, and ***<i>p</i><0.001. Values represent means ± SEM (<i>N</i> = 5). (a) ROS production, (b) Levels of lipid, protein, and DNA peroxidation.</p

Ultrastructure of the brain tissue in fifth-instar larvae after phoxim exposure 48 h.

<p>(a) Control; (b) TiO₂ NPs; (c) Phoxim; (d) TiO₂ NPs + Phoxim. Green arrows indicate karyopyknosis and chromatin marginalization, blue arrows show mitochondria swelling and became deformed, crest broken.</p

Effects of TiO₂ NPs on body weight, survival of phoxim-exposed fifth-instar larvae.

<p>*<i>P</i><0.05, and ***<i>P</i><0.001. Values represent means ± SEM (<i>n</i> = 5).</p

Detection of cathepsin B (CB) activity in Eca-109 cells by the CB probe.

<p>(a) Nuclei of Eca-109 cells which were stained with DAPI (Blue). (b) CB expression in cytoplasm of Eca-109 cells staining with FITC (Green). (c) Fluorescence signals in cytoplasm of Eca-109 cells due to the probe activation (Red). (d) The complete coincidence of CB expression and fluorescence signals in merged image indicating the high specificity of the CB probe to CB activity. Original magnification, 400×.</p

Cathepsin B (CB) expression in human esophageal tissues and two cell lines.

<p>Positive expression was observed in intraepithelial neoplasia (IN) I(b), IN II(c), IN III (d), tumor in situ (e) and esophageal squamous cell carcinoma (f) but not in normal esophageal mucosa (a). (g) Eca-109 cells (human esophageal squamous cell carcinoma cell line) had a high level of CB expression, but Het-1A cells (normal human esophageal epithelial cell line) had no CB expression. β-actin was used as an internal control. Original magnification, 100×.</p