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Intracellular reactive oxygen species (ROS) may participate in cellular responses to various stimuli including hemodynamic forces and act as signal transduction messengers. Human umbilical vein endothelial cells (ECs) were subjected to laminar shear flow with shear stress of 15, 25, or 40 dynes/cm2 in a parallel plate flow chamber to demonstrate the potential role of ROS in shear-induced cellular response. The use of 2',7'-dichlorofluorescin diacetate (DCFH-DA) to measure ROS levels in ECs indicated that shear flow for 15 minutes resulted in a 0.5- to 1.5-fold increase in intracellular ROS. The levels remained elevated under shear flow conditions for 2 hours when compared to unsheared controls. The shear-induced elevation of ROS was blocked by either antioxidant N-acetyl-cysteine (NAC) or catalase. An iron chelator, deferoxamine mesylate, also significantly reduced the ROS elevation. A similar inhibitory effect was seen with a hydroxyl radical (.OH) scavenger, 1,3-dimethyl-2-thiourea (DMTU), suggesting that hydrogen peroxide (H202), .OH, and possibly other ROS molecules in ECs were modulated by shear flow. Concomitantly, a 1.3-fold increase of decomposition of exogenously added H2O2 was observed in extracts from ECs sheared for 60 minutes. This antioxidant activity, abolished by a catalase inhibitor (3-amino-1,2,4-triazole), was primarily due to the catalase. The effect of ROS on intracellular events was examined in c-fos gene expression which was previously shown to be shear inducible. Decreasing ROS levels by antioxidant (NAC or catalase) significantly reduced the induction of c-fos expression in sheared ECs. We demonstrate for the first time that shear force can modulate intracellular ROS levels and antioxidant activity in ECs. Furthermore, the ROS generation is involved in mediating shear-induced c-fos expression. Our study illustrates the importance of ROS in the response and adaptation of ECs to shear flow.

In this study, a novel freeze-gelation method instead of the conventional freeze-drying method was used to fabricate porous chitosan/collagen-based composite scaffolds for skin-related tissue engineering applications. To improve the performance of chitosan/collagen composite scaffolds, we added 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and amino acids (including alanine, glycine, and glutamic acid) in the fabrication procedure of the composite scaffolds, in which amino acid molecules act as crosslinking bridges to enhance the EDC-mediated crosslinking. This novel combination enhanced the tensile strength of the scaffolds from 0.70 N/g for uncrosslinked scaffolds to 2.2 N/g for crosslinked ones; the crosslinked scaffolds also exhibited slower degradation rates. The hydrophilicity of the scaffolds was also significantly enhanced by the addition of amino acids to the scaffolds. Cell compatibility was demonstrated by the in vitro culture of human skin fibroblasts on the scaffolds. The fibroblasts attached and proliferated well on the chitosan/collagen composite scaffolds, especially the one with glutamic acid molecules as crosslinking bridges, whereas cells did not grow on the chitosan scaffolds. Our results suggest that the collagen-modified chitosan scaffolds with glutamic acid molecules as crosslinking bridges are very promising biomaterials for skin-related tissue engineering applications because of their enhanced tensile strength and improved cell compatibility with skin fibroblasts.

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The use of wheat germ agglutinin (WGA), a lectin molecule, to modify chitosan and enhance the cell-biomaterial interaction was examined. The percentage of living fibroblast cells on the surfaces of tissue culture polystyrene (TCPS) control, WGA-modified chitosan, and unmodified chitosan films increased to 99%, 99%, and 85%, respectively, after seeding for 48 h. DNA staining revealed that a portion of fibroblasts cultivated on chitosan films( )were undergoing apoptosis. In contrast, fibroblasts growing on WGA-modified chitosan film surfaces did not show any indication of apoptosis. The number of fibroblast cells was the highest on the WGA-modified chitosan surfaces, followed by the TCPS and unmodified chitosan surfaces. This WGA-mediated enhancement on the fibroblast cell-biomaterial interaction was cell type dependent. Other types of cells may need different lectin molecules for enhanced interaction with biomaterials. Further, the evaluation of the heat shock protein (HSP) mRNA expression indicated that HSP 90 expression was increased in the fibroblast cells cultivated on chitosan films and decreased to basal levels on the WGA-modified chitosan films. Taken together, our data suggest that the use of WGA and other lectin molecules to enhance the cell-biomaterial interaction via oligosaccharide-mediated cell adhesion is a promising way to improve cell adhesion and proliferation, the two key issues in tissue engineering.

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Chitosan was used in this study to form polyelectrolyte complex (PEC) with water-soluble acidic polysaccharides, including gum arabic and pectin. Porous membranes made of gum arabic or pectin only were quiet fragile. After incorporating with chitosan, the usability of the membranes was greatly improved. The results showed that the gum arabic/chitosan composite membranes had detectable tensile stress and elongation capability. Moreover, the pectin/chitosan composite membranes had significantly improved tensile stress and elongation capability. Both of the two composite membranes had greater water uptake capability than the membranes composed of chitosan only. We have demonstrated that chitosan can function as a material stabilizer to maintain the solid status of the acidic polysaccharides and thus improve the performance of these acidic polysaccharides.

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