Protective effect of edaravone against Alzheimer's disease-relevant insults in neuroblastoma N2a cells.

Oxidative stress has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). Thus, antioxidant therapy may represent a promising avenue for the treatment of AD. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent free radical scavenger and has been shown to provide neuroprotection in both animal models of cerebral ischemia and stroke patients. In the present study, we investigated the protective effect of edaravone against AD-relevant insults in neuroblastoma N2a cells and explored the potential mechanisms involved. N2a/Swe.Δ9 cells were used as the AD model cells, which exhibited reduced cell viability, increased apoptosis and oxidative stress as well as decreased mitochondrial membrane potential compared with N2a/Wt cells. All of these phenotypes were significantly reversed by edaravone treatment. Edaravone treatment significantly elevated cell viability, reduced apoptotic rate, attenuated oxidative stress and improved mitochondrial membrane potential in N2a/Swe.Δ9 cells. Furthermore, edaravone treatment inhibited mitochondria-dependent apoptosis pathways in N2a/Swe.Δ9 cells through decreasing the Bax/Bcl-2 ratio, attenuating cytochrome c release and suppressing the activation of caspase-3. These results demonstrate that edaravone provides neuroprotection in an AD-related in vitro model and therefore, may be a potential complement for AD therapy.

Reconstruction and expression of the MRI-contrast protein, ferritin, with recombinant rabies vectors.

Attenuated recombinant rabies vector could be an ideal system for delivery of contrast agent gene for Magnetic Resonance Imaging (MRI) because of its neurotropic nature. In this study, the gene of a biomolecular contrast agent, ferritin, was successfully cloned into two rabies virus vectors, vaccine-based pCTN and street strain-based pNH. Recombinant virus granules were obtained and proved to express ferritin by RT-PCR after transfection of CTN-ferritin and NH-ferritin vector systems in BHK-21 cells. The recovered rabies virus-rCTN-ferritin was of similar ability to rNH-ferritin, which suggests the possibility of application of this safe and effective rabies vector system in delivery of diagnostic or therapeutic genes into the brain.

Metabolites of cerebellar neurons and hippocampal neurons play opposite roles in pathogenesis of Alzheimer's disease.

Metabolites of neural cells, is known to have a significant effect on the normal physiology and function of neurons in brain. However, whether they play a role in pathogenesis of neurodegenerative diseases is unknown. Here, we show that metabolites of neurons play essential role in the pathogenesis of Alzheimer's disease (AD). Firstly, in vivo and in vitro metabolites of cerebellar neurons both significantly induced the expression of Abeta-degrading enzymes in the hippocampus and cerebral cortex and promoted Abeta clearance. Moreover, metabolites of cerebellar neurons significantly reduced brain Abeta levels and reversed cognitive impairments and other AD-like phenotypes of APP/PS1 transgenic mice, in both early and late stages of AD pathology. On the other hand, metabolites of hippocampal neurons reduced the expression of Abeta-degrading enzymes in the cerebellum and caused cerebellar neurodegeneration in APP/PS1 transgenic mice. Thus, we report, for the first time, that metabolites of neurons not only are required for maintaining the normal physiology of neurons but also play essential role in the pathogenesis of AD and may be responsible for the regional-specificity of Abeta deposition and AD pathology.

PPARgamma transcriptionally regulates the expression of insulin-degrading enzyme in primary neurons.

Insulin-degrading enzyme (IDE) is a protease that has been demonstrated to play a key role in degrading both Abeta and insulin and deficient in IDE function is associated with Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2) pathology. However, little is known about the cellular and molecular regulation of IDE expression. Here we show IDE levels are markedly decreased in DM2 patients and positively correlated with the peroxisome proliferator-activated receptor gamma (PPARgamma) levels. Further studies show that PPARgamma plays an important role in regulating IDE expression in rat primary neurons through binding to a functional peroxisome proliferator-response element (PPRE) in IDE promoter and promoting IDE gene transcription. Finally, we demonstrate that PPARgamma participates in the insulin-induced IDE expression in neurons. These results suggest that PPARgamma transcriptionally induces IDE expression which provides a novel mechanism for the use of PPARgamma agonists in both DM2 and AD therapies.

Inhibition of gamma-secretase activity reduces Abeta production, reduces oxidative stress, increases mitochondrial activity and leads to reduced vulnerability to apoptosis: Implications for the treatment of Alzheimer's disease.

It has been argued that gamma-secretase should be considered as a pharmacological target, as there are few mechanism-based experimental and clinical studies on gamma-secretase treatment. In this study, we found that N2a cells bearing APP695 or its Swedish mutant exhibited increased basal levels of ROS, nitric oxide (NO), protein carbonyls, MDA and intracellular calcium, as well as reduced level of the mitochondrial membrane potential and ATP. When the activity of gamma-secretase was inhibited by expression of the D385A PS1 variant, cells (N2a/Swe.D385A) showed reduced basal levels of ROS, nitric oxide (NO), protein carbonyls, MDA and intracellular calcium, as well as increased mitochondrial membrane potential and ATP level. In addition, N2a/Swe.D385A cells showed reduced vulnerability to H(2)O(2)-induced apoptosis. The Bcl-2 and JNK/ERK pathways were proven to be involved in the change of vulnerability to H(2)O(2)-induced apoptosis. Moreover, we discovered that inhibition of gamma-secretase by DAPT would lead to a reduction of ROS levels and stabilization of mitochondrial function in APP (N2a/APP695) and APP Swedish mutant (N2a/APPswe) transfected cells. At last, it was shown that Abeta antibody and antiserum prevented increase of ROS and reduction of mitochondrial membrane potential in N2a/Swe.DeltaE9 cells but not in N2a/Swe.D385A cells, which indicated that reduced formation of Abeta was the reason for reduction of ROS formation and increase of mitochondrial membrane potential when PS-1 activity was impaired in N2a/Swe.D385A cells. We concluded that neurotoxicity was positively correlated with the activity of gamma-secretase, which suggested inhibition of gamma-secretase is a rational pharmacological target for Alzheimer's disease treatment.

Effect of amyloid beta on capacitive calcium entry in neural 2a cells.

We studied the direct role of amyloid beta (Abeta) in regulating capacitive calcium entry (CCE), an important refilling mechanism for depleted intracellular calcium stores. For the first time, we found that Abeta can potentiate CCE. Neural 2a cells stably expressing Swedish mutant APP (APPswe), which can secrete large amounts of Abeta, have stronger CCE than its wild-type controls. Either reducing the Abeta in the medium by antibody binding or decreasing Abeta production by gamma secretase inhibitor treatment could significantly depress CCE in APPswe cells. The results demonstrated that the CCE potentiation in APPswe cells was caused by Abeta over-expression. Our research also revealed that the effect of Abeta on CCE potentiation could be decreased by Abeta channel blocker, which showed that the channels formed by Abeta are one of the ways through which Abeta causes CCE potentiation.

Abnormal cleavage of APP impairs its functions in cell adhesion and migration.

Amyloid precursor protein (APP) is expressed ubiquitously but its wrong cleavage only occurs in central nervous system. In this research, overexpression of wild type human APP695 was found to stimulate the adhesion and migration of N2a cells. In the cells co-transfected by familial Alzheimer's disease (FAD)-linked Swedish mutant of APP695 gene plus big up tri, openE9 deleted presenilin1 gene (N2a/Swe. big up tri, open9), however, this stimulating function was impaired compared to that in the cells co-transfected by Swedish mutant of APP695 gene plus dominant negative mutant of presenilin1 D385A gene (N2a/Swe.385). Furthermore, it was also found that the phosphorylation of FAK Tyr-861 and GSK-3beta Ser-9 was reduced in N2a/Swe.Delta9 cells, which can be possibly taken as a reasonable explanation for the underlying mechanism. Our results suggest that impaired cell adhesion and migration induced by abnormal cleavage of APP could contribute to the pathological effects in FAD brain.

The association of GSK3 beta with E2F1 facilitates nerve growth factor-induced neural cell differentiation.

It is widely acknowledged that E2F1 and GSK3beta are both involved in the process of cell differentiation. However, the relationship between E2F1 and GSK3beta in cell differentiation has yet to be discovered. Here, we provide evidence that in the differentiation of PC12 cells induced by nerve growth factor (NGF), GSK3beta was increased at both the mRNA and protein levels, whereas E2F1 at these two levels was decreased. Both wild-type GSK3beta and its kinase-defective mutant GSK3beta KM can inhibit E2F1 by promoting its ubiquitination through physical interaction. In addition, the colocalization of GSK3beta and E2F1 and their subcellular distribution, regulated by NGF, were observed in the process of PC12 differentiation. At the tissue level, GSK3beta colocalized and interacted with E2F1 in mouse hippocampus. Furthermore, GSK3beta facilitated neurite outgrowth by rescuing the promoter activities of Cdk inhibitors p21 and p15 from the inhibition caused by E2F1. To summarize, our findings suggest that GSK3beta can promote the ubiquitination of E2F1 via physical interaction and thus inhibit its transcription activity in a kinase activity independent manner, which plays an important role in the NGF-induced PC12 differentiation.

LEF-1 activates the transcription of E2F1.

LEF-1 and E2F are both transcription factors involved in cell proliferation, differentiation and apoptosis. The present study shows for the first time that LEF-1 associates with E2F1 and further beta-catenin independently activates the E2F-responsive reporter gene by attenuating the interaction between E2F1 and Histone deacetylase 1 (HDAC1), which indicates that LEF-1, except for its function in Wnt signaling, may play a distinct role via activating the transcription of E2F1.

Physical properties and biocompatibility of a porous chitosan-based fiber-reinforced conduit for nerve regeneration.

Porous fiber-reinforced chitosan nerve conduits were fabricated from chitosan yarns and a chitosan solution by combining an industrial braiding method with a mold casting/lyophilization technique. The conduits were permeable to molecules ranging in molecular size from 180 Da (glucose) to 66,200 Da (BSA). The compressive load of the reinforced conduits was significantly higher than that of a non-reinforced control conduit at equal levels of strain. The tensile strength of the reinforced conduits was also increased from 0.41 +/- 0.17 to 3.69 +/- 0.64 MPa. An in vitro cytotoxicity test showed the conduits were not cytotoxic to Neuro-2a cells. Preliminary in vivo implantation testing indicated that the conduits were compatible with the surrounding tissue.

Biomimetic coating of compound titania and hydroxyapatite on titanium.

The modification on the titanium implant surface is an effective method to improve the biocompatibility of titanium. This article describes efforts to improve implant biocompatibility by applying titania and hydroxyapatite to form a three-layer coating on the titanium surface. This three-layer coating is made up of HA as the top layer (formed by hydrothermal treatment), porous TiO2 as the middle layer (formed by micro-arc oxidation) and a dense TiO2 film as the inner layer (formed by preanodic oxidation). The physicochemical characteristics, cell behavior and in vivo studies were assessed. The physicochemical characteristics were investigated using scanning electron micoscopy observation, fibronectin and laminin adsorption, corrosion test and X-ray diffraction analysis. Cell behavior included morphology observation with scanning electron microscopy (SEM), number count with methylthiazol tetrazolium (MTT) assay and Alkaline phosphatase (ALP, a representative enzyme of osteoblastic differentiation) activity of osteoblast-like MC3T3-E1 cells. In study in vivo the specimens were embedded in skull wound for repair. By the analysis of experiments, the titanium coated with this three-layer coating has been proved to have excellent corrosion resistance and good biocompatibility, which can promote cell proliferation and bone formation. So this modified titanium is an improved alternative to untreated titanium for bone repair applications.

[Proliferation and differentiation of MC 3T3-E1 cells cultured on nanohydroxyapatite/chitosan composite scaffolds].

with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 3T3-E1 the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTZ assay. After being Abstract Nanohydroxyapatite/chitosan composite scaffolds were fabricated and the proliferation and differentiation of preosteoblast MC 3T3-E1 on them were examined for the assessment of their biocompatibility. Nanohydroxyapatite was combined with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 333-E1 cells were inoculated into the porous composite scaffolds and chitosan scaffolds, respectively. The morphology of cells cultured on the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTT assay. After being cultured in conditioned medium for 30 days, the cells' alkaline phosphatase activities on the scaffolds were studied in situ to compare their differentiation levelabout. Moreover, the alkaline phosphatase activities were assessed with a kit. The expression level of characteristic osteogenic gene was evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The results indicated that MC 3T3-E1 cells grown on the composite scaffolds showed a higher proliferation rate and spread better than that on chitosan scaffolds. The alkaline phosphatase stain results showed that the alkaline phosphatase activity of cells on composite scaffolds was significantly higher than that on the chitosan scaffolds. In addition, the quantitative examination of alkaline phosphatase activity indicated that the cells cultured on the composite scaffolds expressed an activity level about 8 times higher than that on chitosan scaffolds. Simultaneously, the osteogenic gene osteopontin (OPN) of cells cultured on composite scaffolds showed a higher expression level than that on chitosan scaffolds. Another osteogenic gene osteocalcin (OC) was expressed in cells cultured on composite scaffolds, whereas it was not detected in cells on chitosan scaffolds. The addition of nanohydroxyapatite in the scaffolds improved not only the proliferation but also the differentiation of preosteoblast cultured on them. The composite scaffolds showed good biocompatibility and bioactivity. These scaffolds would be promising in bone tissue engineering.

Preparation and characterization of a multilayer biomimetic scaffold for bone tissue engineering.

In scaffold based bone tissue engineering, both the pore size and the mechanical properties of the scaffold are of great importance. However, an increase in pore size is generally accompanied by a decrease in mechanical properties. In order to achieve both suitable mechanical properties and porosity, a multilayer scaffold is designed to mimic the structure of cancellous bone and cortical bone. A porous nano-hydroxyapatite-chitosan composite scaffold with a multilayer structure is fabricated and encased in a smooth compact chitosan membrane layer to prevent fibrous tissue ingrowth. The exterior tube is shown to have a small pore size (15-40 microm in diameter) for the enhancement of mechanical properties, while the core of the multilayer scaffold has a large pore size (predominantly 70-150 microm in diameter) for nutrition supply and bone formation. Compared with the uniform porous scaffold, the multilayer scaffold with the same size shows an enhanced mechanical strength and larger pore size in the center. More cells are shown to grow into the center of the multilayer scaffold in vitro than into the uniform porous scaffold under the same seeding condition. Finally, the scaffolds are implanted into a rabbit fibula defect to evaluate the osteoconductivity of the scaffold and the efficacy of the scaffold as a barrier to fibrous tissue ingrowth. At 12 weeks post operation, affluent blood vessels and bone formation are found in the center of the scaffold and little fibrous tissue is noted in the defect site.

Characterization and osteoblast-like cell compatibility of porous scaffolds: bovine hydroxyapatite and novel hydroxyapatite artificial bone.

Three different porous scaffolds were tested. The first two were prepared by sintering bovine bone. The third scaffold was prepared using three-dimensional gel-lamination, a new rapid prototyping method, and was named as hydroxyapatite artificial bone. X-ray diffraction and Fourier transform infrared spectroscopy analysis confirmed that the samples were mainly highly crystalline hydroxyapatite ceramics. Scanning electron microscopy and mercury intrusion porosimetry measurement showed that the pores were interconnected and pore sizes ranged from several microns to hundreds of microns. Mouse osteoblast-like cells grown on the three scaffolds retained their characteristic morphology. Cell proliferation and differentiation, analyzed by methylthiazol tetrazolium (MTT) and alkaline phosphatase activity assays, were significantly higher on the hydroxyapatite artificial bone than on the other two scaffolds tested. All the scaffolds provided good attachment, proliferation and differentiation of bone cells. These results indicate that the scaffolds have a favorable interaction with cells, they support cell growth and functions, and therefore these scaffolds may have great potential as bone substitutes. The three-dimensional gel-lamination method is proven to be an attractive process to design and fabricate bone scaffolds with favorable properties, and therefore, has promising potential for bone repair applications.

Corrosion test, cell behavior test, and in vivo study of gradient TiO2 layers produced by compound electrochemical oxidation.

This paper describes efforts to improve implant biocompatibility and durability by applying a hybrid technique using composite oxidation. Pure titanium was used as the substrate material. A porous oxide film as the outer layer was produced by micro-arc oxidation and a dense oxide film as the inner layer was produced by pre-anodic oxidation. In this study, physicochemical characteristics, corrosion test, cell attachment behavior, and in vivo studies were used to compare this gradient layer with untreated titanium. The results revealed that the gradient layer was composed of two layers of oxide films which were made up of rutile and anatase and the surface was porous with calcium and phosphor. The corrosion resistance of the gradient layer was improved remarkably, which was about three times the values for titanium and two times the value for the dense layer. The cell-material interaction study indicated that L929 cells seeded and cultured on the gradient layer appeared to attach well and the rate of proliferation was the greatest. The study in vivo showed that the gradient layer had good biocompatibility. This gradient layer provides a material with high corrosion resistance, bioactivity, and biological properties suitable for tissue engineering applications.

Investigation of MC3T3-E1 cell behavior on the surface of GRGDS-coupled chitosan.

The GRGDS (Gly-Arg-Gly-Asp-Ser) peptide has intermediate affinity to alphaVbeta3 and alphaIIbbeta3, which are the integrins most reported to be involved in bone function. In this study, biomimetic chitosan films modified with GRGDS peptide were prepared and were used as a substrate for the in vitro culture of MC3T3-E1 cells in order to investigate the effect of GRGDS modification on MC3T3-E1 cell behavior. The results of electron spectroscopy for chemical analysis (ESCA), attenuated total reflection-Fourier transform infrared spectra (ATR-FTIR), and amino acid analysis (AAA) demonstrated that the chitosan films were successfully modified with GRGDS peptides and that the surface density of the immobilized GRGDS was on the order of 10(-9) mol/cm2. The immobilization of the GRGDS sequence on chitosan as well as the peptide concentration play a significant role in MC3T3-E1 cell behavior. MC3T3-E1 cell attachment, proliferation, migration, differentiation, and mineralization were remarkably greater on GRGDS-coupled chitosan than on unmodified chitosan. Besides, the degree of acceleration of these biological processes was found to be dependent on peptide density. Competitive inhibition of MC3T3-E1 cell attachment using soluble GRGDS peptides indicated that the interaction of MC3T3-E1 cells with the surface of the materials was ligand-specific. Cytoskeleton organization in the fully spread MC3T3-E1 cells was highly obvious on GRGDS-coupled chitosan when compared to the lack of actin fibers noted in the round MC3T3-E1 cells on unmodified chitosan. These results suggest that MC3T3-E1 cell function can be modulated, in a peptide density-dependent manner, by the immobilization of GRGDS peptide on chitosan used for scaffold-based bone tissue engineering.

A sandwich tubular scaffold derived from chitosan for blood vessel tissue engineering.

Many materials have been investigated in blood vessel tissue engineering, such as PGA, PLGA, P4HB. However, chitosan is not mentioned in the arena. This study aimed to develop a chitosan-based tubular scaffold and examine its feasibility of being applied in this field. Briefly, a knitted chitosan tube was dipped into chitosan solution (2%, w/v) and dried, then its inner and outer surface was mantled with a layer of chitosan/gelatin (4:1, w/w) complex solution, and then freeze-dehydrated. In vitro characterization showed that the scaffold had a wall of 1.0 mm in thickness with a sandwich structure, and a porosity of 81.2%. The pore diameter was 50-150 microm and could be regulated by varying freezing conditions. The scaffold possessed proper swelling property, burst strength of almost 4000 mmHg, and high suture-retention strength. After degradation for 2 months, the scaffold could maintain enough mechanical strength with an average mass loss of 18.7%. Vascular smooth muscle cells could spread and grow very well on the scaffold. This study provided a novel method to fabricate chitosan and its complex into a tubular scaffold and demonstrated the feasibility of the scaffold employed in the field of blood vessel tissue engineering.

Novel biodegradable films and scaffolds of chitosan blended with poly(3-hydroxybutyrate).

In order to develop a novel biomaterial, films of chitosan blended with poly(3-hydroxybutyrate) (PHB) were prepared by an emulsion blending technique and their properties were characterized. Scanning electron microscopy (SEM) showed that PHB microspheres were formed and were entrapped in chitosan matrices, which made the film surface rough. With increasing PHB content, the roughness of the film surface increased, while the swelling capability of the films decreased. In a wet state, the blended films exhibited a lower elastic modulus, a higher elongation-at-break and a higher tensile strength compared with chitosan films. Cell-culture experiments revealed that the blended films had better cytocompatibility than chitosan films. To explore the potential application of the blended material in tissue engineering, the porous blended scaffolds were fabricated and their pore morphology was observed by SEM. The results revealed that not only pore structure but also pore wall morphology of the blended scaffolds could be controlled by selecting the parameters of the fabrication process. These advantageous properties indicate that the blended chitosan/PHB material is promising for tissue engineering applications.