Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells.

Laminar shear flow triggers a signaling cascade that maintains the integrity of endothelial cells (ECs). Hydrogen sulfide (H2S), a new gasotransmitter is regarded as an upstream regulator of nitric oxide (NO). Whether the H2S-generating enzymes are correlated to the enzymes involved in NO production under shear flow conditions remains unclear as yet. In the present study, the cultured ECs were subjected to a constant shear flow (12 dyn/cm(2)) in a parallel flow chamber system. We investigated the expression of three key enzymes for H2S biosynthesis, cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS), and 3-mercapto-sulfurtransferase (3-MST). Shear flow markedly increased the level of 3-MST. Shear flow enhanced the production of H2S was determined by NBD-SCN reagent that can bind to cysteine/homocystein. Exogenous treatment of NaHS that can release gaseous H2S, ECs showed an increase of phosphorylation in Akt(S473), ERK(T202/Y204) and eNOS(S1177). This indicated that H2S can trigger the NO-production signaling cascade. Silencing of CSE, CBS and 3-MST genes by siRNA separately attenuated the phosphorylation levels of Akt(S473) and eNOS(S1177) under shear flow conditions. The particular mode of shear flow increased H2S production. The interplay between H2S and NO-generating enzymes were discussed in the present study.

Use of gum arabic to improve the fabrication of chitosan-gelatin-based nanofibers for tissue engineering.

Current techniques for fabricating chitosan-gelatin-based nanofibers require the use of corrosive and expensive solvents. Our novel method, however, using gum arabic and a mild (20 wt%) aqueous acetic acid solution as solvent can produce a solution with much higher chitosan-gelatin content (16 wt%). Without gum arabic, which greatly decreases the viscosity of the solution, such an outcome was unachievable. The solution was utilized to prepare electrospun chitosan-gelatin-polyvinyl alcohol-gum arabic nanofibers with a weight ratio of 8:8:2:0.5 (C8G8P2A0.5 nanofibers), in which polyvinyl alcohol could stabilize the electrospinning process. The stability and tensile strength (2.53 MPa) of C8G8P2A0.5 nanofibers (mats) were enhanced by glutaraldehyde crosslinking. Furthermore, mesenchymal stem cells attached and proliferated well on the mat. The strength-enhanced and cytocompatible C8G8P2A0.5 mats are thereby suitable for tissue engineering applications. More importantly, we have created a less expensive and safer method (one not using hazardous solvents) to fabricate chitosan-gelatin-based nanofibers.

Shrinking mechanism of a porous collagen matrix immersed in solution.

The porous structure of collagen-based matrices enables the infiltration of cells both in in vitro and clinical applications. Reconstituted porous collagen matrices often collapse when they are in contact with aqueous solutions; however, the mechanism for the collapse of the pores is not understood. We, therefore, investigated the interactions between the collagen matrix and different solutions, and discuss the mechanisms for the change in microstructure of the matrix on immersing it in solution. When a dried collagen matrix was immersed in aqueous solutions, the matrix shrunk and pores close to the surface closed. The shrinkage ratio and thickness of the compact microstructure close to the superficial area decreased with increasing ethanol content in the solution. The original porous structure of the collagen matrix was preserved when the matrix was immersed in absolute ethanol. The shrinkage of a porous collagen matrix in contact with aqueous solutions was attributed to the liquid/gas interfacial tension. The average pore diameter of the matrix also significantly affected the shrinkage of the matrix. The shrinkage of the matrix, explained using the Young-Laplace equation, was found to result from the pressure drop, and especially in the pores located superficially, leading to the collapse of the matrix microstructure. The integrity of the porous microstructure allows better penetration of cells in medical applications. The numbers of NIH/3T3 fibroblasts penetrated through the hydrated Col/PBS porous collagen matrices pre-immersed in absolute ethanol with subsequent water and DMEM culture medium replacements were significantly higher than those through matrices hydrated directly in DMEM.

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications.

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.

Cellulose-based diagnostic devices for diagnosing serotype-2 dengue fever in human serum.

Here, two types of cellulose-based in vitro diagnostic devices are demonstrated for the diagnosis of dengue virus infection in both buffer system and human serum: 1) paper-based ELISA for providing the semiquantitative information of the disease activity of serotype-2 dengue fever to healthcare persons (i.e., monitoring the disease activity with a specific serotype in single patients); 2) lateral flow immunoassays to screen for infection with serotype-2 dengue fever (i.e., rapid YES or NO diagnosis prepared for large populations, in terms of global public health). Paper-based ELISA (specific to serotype-2 dengue fever), which builds off of our previous studies and a revised previous ELISA procedure, owns multiple advantages: 1) high sensitivity (about 40 times higher than the current ELISA-based approaches, due to our therapeutic-based monoclonal antibody) and specificity (specific to dengue virus serotype-2 nonstructural protein-1 antigens); 2) tiny amount of sample and reagent used for single tests; 3) short operating duration (i.e., rapid diagnostic device); and, 4) inexpensiveness (appropriate for use in all developing and underdeveloped nations of the world). Due to the higher sensitivity and shorter operating duration of paper-based ELISA (compared with conventional ELISA, and lateral flow immunoassays also performed in this study), this study has not only been able to perform the diagnosis of dengue virus serotype-2 nonstructural protein-1 antigens in both buffer system and human serum but also to evaluate dengue virus serotype-2 envelope proteins in the buffer system, thus successfully achieving the first such use of these proteins as the target antigen for the development of diagnostic tools. These results provide a more comprehensive understanding for the genesis of dengue fever diagnostic tools (through antibody-antigen recognition).

Chitosan/pectin/gum Arabic polyelectrolyte complex: process-dependent appearance, microstructure analysis and its application.

Novel chitosan/pectin/gum Arabic polyelectrolyte complex (PEC) solutions and membranes with various compositions were prepared for biomedical applications. The appearance of the PEC solutions, either clear or turbid, was process-dependent and depended on how the three components were dissolved and mixed. The addition of gum Arabic to the chitosan and pectin significantly decreased the viscosities of the resultant PEC solutions due to the formation of globe-like microstructures that was accompanied by network-like microstructures and other molecular entanglements. The mechanical strength and hydrophilicity of the PEC membranes manufactured from the PEC solutions, especially for a weight ratio of 84/8/8 (chitosan/pectin/gum Arabic), were enhanced compared to pure chitosan membranes. Moreover, the use of the 84/8/8 PEC membranes as a drug carrier exhibited steady and fairly complete release of a drug (insulin) for 6h. Based on these promising results, the chitosan/pectin/gum Arabic PEC membranes have great potential in controlled drug release applications.

Interactions between chitosan and cells measured by AFM.

Chitosan, a biocompatible material that has been widely used in bone tissue engineering, is believed to have a high affinity to osteoblastic cells. This research is the first to prove this hypothesis. By using atomic force microscopy (AFM) with a chitosan-modified cantilever, quantitative evaluation of the interforce between chitosan and cells was carried out. A chitosan tip functionalized with Arg-Gly-Asp (RGD) was also used to measure the interforce between RGD-chitosan and osteoblastic cells. This research concluded by examining cell adhesion and spreading of chitosan substrates as further characterization of the interactions between cells and chitosan. The force measured by AFM showed that the interforce between chitosan and osteoblasts was the highest (209 nN). The smallest adhesion force (61.8 nN) appeared between chitosan and muscle fibroblasts, which did not demonstrate any osteoblastic properties. This result proved that there was a significant interaction between chitosan and bone cells, and correlated with the observations of cell attachment and spreading. The technique developed in this research directly quantified the adhesion between chitosan and cells. This is the first study to demonstrate that specific interaction exists between chitosan and osteoblasts.

Regulation of shear-induced nuclear translocation of the Nrf2 transcription factor in endothelial cells.

BACKGROUND: Vascular endothelial cells (ECs) constantly experience fluid shear stresses generated by blood flow. Laminar flow is known to produce atheroprotective effects on ECs. Nrf2 is a transcription factor that is essential for the antioxidant response element (ARE)-mediated induction of genes such as heme-oxygenase 1 (HO-1). We previously showed that fluid shear stress increases intracellular reactive oxygen species (ROS) in ECs. Moreover, oxidants are known to stimulate Nrf2. We thus examined the regulation of Nrf2 in cultured human ECs by shear stress. RESULTS: Exposure of human umbilical vein endothelial cells (HUVECs) to laminar shear stress (12 dyne/cm2) induced Nrf2 nuclear translocation, which was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, a protein kinase C (PKC) inhibitor, and an antioxidant agent N-acetyl cysteine (NAC), but not by other protein kinase inhibitors. Therefore, PI3K, PKC, and ROS are involved in the signaling pathway that leads to the shear-induced nuclear translocation of Nrf2. We also found that shear stress increased the ARE-binding activity of Nrf2 and the downstream expression of HO-1. CONCLUSION: Our data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system.

Use of dicarboxylic acids to improve and diversify the material properties of porous chitosan membranes.

Several nontoxic dicarboxylic acid solutions (oxalic acid, succinic acid, malic acid, and adipic acid solutions) instead of an acetic acid solution were used as solvents for chitosan dissolution. The amount of free amino groups of the chitosan in the solution decreased due to the ionic cross-linking of the dicarboxylic acids with chitosan. These solutions were used to fabricate porous chitosan membranes. Replacing acetic acid with these dicarboxylic acids for membrane preparation improved the water uptake (by 35% at most), tensile strength (by 110% at most), and elongation capability (by 50% at most) of the membranes. These dicarboxylic acid solutions not only act as solvents but also improve the material properties of the chitosan membranes due to the ionic cross-linking and hydrogen bond formation. In brief, a nontoxic and straightforward cross-linking method has been developed for chitosan material; this method does not result in a brittle product, thus making it better than the use of toxic cross-linking reagents.

Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells.

For long-term culture, murine adipose-derived stromal cells (mADSCs) at latter passages demonstrated a marked decline in proliferative activity, exhibited senescent morphology and reduced differentiation potentials, particularly osteogenesis. To extend the lifespan of mADSCs, two culture conditions containing hyaluronan (HA) was compared in our study, one as a culture medium supplement (SHA), and the other where HA was pre-coated on culture surface (CHA). mADSCs cultivated with SHA exhibited a prolonged lifespan, reduced cellular senescence, and enhanced osteogenic potential compared to regular culture condition (control). Upon CHA treatment, mADSCs tended to form cell aggregates with gradual growth profiles, while their differentiation activities remained similar to SHA groups. After transferring mADSCs from CHA to control surface, they were shown to have an extended lifespan and an increase of osteogenic potential. Our results suggested that HA can be useful for preserving the proliferation and differentiation potentials of long-term cultured mADSCs.

Efficient modification on PLLA by ozone treatment for biomedical applications.

RGDS (Arg-Gly-Asp-Ser) is immobilized on poly(L-lactic acid) (PLLA) with ozone oxidation and the addition of an intermediate reactant, acryl succinimide (ASI) to promote the grafting efficiency. A DPPH (2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl) assay has revealed that the peroxide concentration can be controlled by adjusting the ozone treatment time. The immobilization of ASI is verified by elemental analysis. The peptide concentrations are in the effective order, as shown by means of high performance liquid chromatography (HPLC), and the grafting efficiency is proven to be relatively high compared with the previous studies. The culture of rat osteosarcoma 17/2.8 (ROS), osteoblastic-like cells, demonstrates that the grafting of RGDS can enhance the attachment and osteogenesis of ROS cells on PLLA. With the addition of ASI, the cultured ROS cells express normal function in proliferation and mineralization. From in vivo experiments, ASI immobilized on the surface is shown to be biocompatible. These results lead to the conclusion that the ozone treatment with the intermediate reactant ASI is an efficient, biocompatible, and easily controllable procedure to modify PLLA. Furthermore, the immobilization of RGDS in significant amounts following the ozone oxidation could further promote the biocompatibility and the osteoinduction of PLLA.

Laminar flow attenuates interferon-induced inflammatory responses in endothelial cells.

OBJECTIVE: Atherosclerosis is a chronic disease that involves inflammation, in which cytokines, including interferon-gamma (IFNgamma), participate. Endothelial cells (ECs) exposed to IFNgamma increase the expression of CXC chemokines. ECs subjected to laminar flow (LF) are atheroprotective, despite an unclear mechanism. This study was conducted to analyze whether ECs under LF were protected from IFNgamma-induced responses. METHODS: IFNgamma-treated human umbilical cord ECs were subjected to LF in a well-defined flow chamber system. IFNgamma-induced STAT1 activation and downstream target genes were examined. RESULTS: ECs exposed to IFNgamma triggered STAT1 activation via the phosphorylation of Tyr701 and Ser727 in STAT1. ECs exposed to LF alone did not activate STAT1. LF exposure of IFNgamma-treated ECs significantly attenuated IFNgamma-induced Tyr701 phosphorylation in a shear-force- and time-dependent manner, whereas Ser727 phosphorylation was unaffected. Consistently, LF inhibited IFNgamma-induced STAT1 binding to DNA. ECs treated with IFNgamma induced the expression of three T-cell-specific CXC chemokines (CXCL9, CXCL10 and CXCL11) as well as CIITA, a transcriptional regulator of major histocompatibility complex class II (MHCII). Consistently, LF exposure of IFNgamma-treated ECs reduced the expression of CXC chemokines and CIITA. CONCLUSIONS: LF attenuates IFNgamma-induced responses via the suppression of STAT1 activation. Inhibition by LF of the interferon-induced ECs' response may explain some aspects of LF's atheroprotective effects on the endothelium.

Refolding of partially and fully denatured lysozymes.

Lysozyme refolding with high yields sometimes results from incomplete denaturation. Dithiothreitol (DTT) is a reductant commonly used to reduce and unfold disulfide-stabilized lysozymes. Through the use of fluorescence spectroscopy to access the extent of denaturation, we found that the rate and extent of denaturation highly depended on the concentration of DTT. Further, the denaturation exhibited a two-phase transition at a high DTT concentration with DTT at >100 mM and long denaturation time (>24 h) being needed for complete denaturation. A low DTT concentration and a short denaturation time resulted in fast refolding with high activity recovery, while a high DTT concentration and a long denaturation time resulted in slow refolding with low activity recovery. Hence, the renaturation of disulfide-containing lysozyme was highly affected by the extent of denaturation.

Promotion of cell affinity of porous PLLA scaffolds by immobilization of RGD peptides via plasma treatment.

In the present work, RGDS (Arg-Gly-Asp-Ser) was immobilized on PLLA scaffolds with plasma treatment. The amount of immobilization, determined by HPLC, was confirmed to be in the effective order. Results from the culture of rat osteosarcoma (ROS), osteoblastic-like cells, demonstrate that the immobilization of RGDS could effectively enhance the attachment of ROS cells on PLLA and increase the cell density in PLLA scaffolds. In addition, experiments of in vitro mineralization indicate that there were more cells and mineralization focci in the RGDS-immobilized scaffolds, suggesting a tendency to form bone-like tissues, compared with the unmodified PLLA scaffold. On the other hand, the PLLA scaffolds immobilized with RGES (Arg-Gly-Glu-Ser) were much less effective in promotion of ROS attachment, suggesting that the enhancement on cell attachment was mainly due to the recognition of RGDS by the adhesion receptors on the cell membrane. The results presented in this work demonstrate that RGDS could be successfully immobilized on PLLA scaffolds with plasma treatment and such modification can make PLLA scaffolds more suitable for culture of osteoblast-like cells and for generation of bone-like tissues.

Fabrication and release behavior of a novel freeze-gelled chitosan/gamma-PGA scaffold as a carrier for rhBMP-2.

OBJECTIVE: The aim of this study was to fabricate a novel composite porous scaffold by blending chitosan and gamma-poly(glutamic acid) (gamma-PGA) for the sustained delivery of rhBMP-2. METHODS: Chitosan and gamma-PGA were blended to fabricate a novel porous scaffold by the freeze-gelation method. For comparison, scaffolds made of freeze-dried chitosan, freeze-dried PLLA, and freeze-gelled chitosan were also prepared. The scaffolds were loaded with rhBMP-2, and then the controlled release of rhBMP-2 from the scaffolds was assessed by ELISA. RESULTS: The freeze-gelled chitosan/gamma-PGA scaffold (M0=318.29 ng, k=0.32 d(-1)) gave the most satisfactory release curve, followed by the freeze-gelled chitosan (M0=392.76 ng, k=0.59 d(-1)), freeze-dried chitosan (M0=229.21 ng, k=2.28 d(-1)), and freeze-dried PLLA (M0=8.4 ng, k=482.54 d(-1)) scaffolds. In the stability test, p-dioxane (the solvent for PLLA) seriously deteriorated rhBMP-2, whereas acetic acid (the solvent for chitosan) did not. SIGNIFICANCE: A novel chitosan/gamma-PGA composite scaffold for the controlled release of rhBMP-2 was established, with an enhanced release amount and sustained release behavior. This scaffold has many potential applications in bone regenerative therapies.

Polyglycolic acid/chitosan glue and apoptosis of endometriotic cells.

OBJECTIVE: To induce apoptosis of endometriotic cells of patients with endometriosis. DESIGN: To demonstrate that polyglycolic acid/chitosan glue directly inhibits cell proliferation by inducing apoptosis. SETTING: University hospital infertility center. PATIENT(S): Twelve women who visited the center for infertility therapy. INTERVENTION(S): Polyglycolic acid/chitosan glue was applied into primary endometriotic cells; the manipulated cells were collected 1-4 days after polyglycolic acid/chitosan glue treatment. MAIN OUTCOME MEASURE(S): Primary endometriotic cell cultures from eutopic endometriotic tissue were established. The effect of the novel biological glue, polyglycolic acid/chitosan glue A, on endometrial cells in vitro was examined. The different stages of apoptosis were analyzed using flow cytometry with fluorescein isothiocyanate conjugate (FITC)-annexin V and propidium iodide staining. RESULT(S): The growth inhibitory effects of polyglycolic acid/chitosan glue A on endometrial cells were found to be dose-response and time dependent. Less than 15% viability was detected in cultures containing 2,000 microg of polyglycolic acid/chitosan glue A after 4 days of treatment. Induced apoptosis and caspase activity were revealed. The caspase-3 activity increased 2.2-fold with 4 days of culture with 2,000 microg of polyglycolic acid/chitosan glue A. CONCLUSION(S): This is the first study to demonstrate that polyglycolic acid/chitosan glue directly inhibits cell proliferation by inducing apoptosis, thus suggesting that this new biological glue may be useful for endometriosis therapy.

Preparation of gamma-PGA/chitosan composite tissue engineering matrices.

Gamma-poly(glutamic acid) (gamma-PGA), a hydrophilic and biodegradable polymer, was chosen to modify chitosan matrices to produce a gamma-PGA/chitosan composite biomaterial. Three types of both dense and porous composite matrices containing different amounts of gamma-PGA were fabricated. Chitosan and gamma-PGA matrices were also prepared as controls. Fluorescence staining indicated that chitosan and gamma-PGA were evenly distributed in the composite matrices. SEM micrographs showed that an interconnected porous structure with a pore size of 30-100 microm was present in all porous matrices except the gamma-PGA ones. By increasing the percentage of gamma-PGA from 0% to 20%, the swelling ratio of the matrices was enhanced from 1.6 to 3.2. Similarly, the contact angle of the matrices decreased from 113 degrees to 94 degrees . These data suggested that the surface hydrophilicity, water absorption rate, and swelling ratio were improved by adding gamma-PGA to the matrices. Additionally, the mechanical strength of the porous gamma-PGA/chitosan matrices was about 25-50%, higher than that of the unmodified chitosan matrices. The composite matrices were also examined and found to be an appropriate environment for cell attachment and proliferation. The cell density on the 20% gamma-PGA-modified matrices was almost triple that on the unmodified chitosan matrices on day 5. In summary, the gamma-PGA/chitosan composite matrices, due to their better hydrophilic, cytocompatible, and mechanical properties, are very promising biomaterials for tissue engineering applications.

Preparation and characterization of RGD-immobilized chitosan scaffolds.

Chitosan scaffolds were modified with RGDS (Arg-Gly-Asp-Ser) in the present work via an imide-bond forming reaction between amino groups in chitosan and carboxyl groups in peptides. Successful immobilization was verified with FTIR spectroscopy, and the immobilized amount was determined to be on the order of 10(-12) mol/cm2 through analysis of the immobilized amino acids. Results of experiments of cell culture with rat osteosarcoma (ROS) cells demonstrated that RGDS immobilization could enhance the attachment of ROS cells onto the chitosan, resulting in higher cell density attached to the RGDS-modified scaffold than to the unmodified scaffold. It should be noted that only RGDS, but not other peptide such as RGES, is effective in enhancing cell attachment and possible proliferation. Experiments of in vitro mineralization indicated that there were more cells on the RGDS-modified scaffold than on the unmodified scaffold, which tended to form bone-like tissues. The results presented in this work suggest that immobilization of RGDS can make chitosan scaffolds more compatible for the culture of osteoblast-like cells and the regeneration of bone-like tissues.

Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow.

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-l-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G(2)/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses.

Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods.

Freeze-fixation and freeze-gelation methods are presented in this paper which can be used to prepare highly porous scaffolds without using the time and energy consuming freeze-drying process. The porous structure was generated during the freeze of a polymer solution, following which either the solvent was extracted by a non-solvent or the polymer was gelled under the freezing condition; thus, the porous structure would not be destructed during the subsequent drying stage. Compared with the freeze-drying method, the presented methods are time and energy-saving, with less residual solvent, and easier to be scaled up. Besides, the problem of formation of surface skin can be resolved and the limitation of using solvent with low boiling point can be lifted by the presented methods. With the freeze-extraction and freeze-gelation methods, porous PLLA, PLGA, chitosan and alginate scaffolds were successfully fabricated. In addition to the presentation of the morphologies of the fabricated scaffolds, preliminary data of cell culture on them are as well included in the present work.