Type I collagen is a favorable substrate for cell adhesion and growth and is remodelable by many tissue cells; these characteristics make it an attractive material for the study of dynamic cellular processes. Low mass fraction (1.0-3.0 mg/ml), hydrated collagen matrices used for threedimensional cell culture permit cellular movement and remodeling, but their microstructure and mechanics fail to mimic characteristics of many extracellular matrices in vivo and limit the definition of fine-scale geometrical features (< 1 mm) within scaffolds. In this study, we worked with hydrated type I collagen at mass fractions between 3.0 and 20 mg/ml to define the range of densities over which the matrices support both microfabrication and cellular remodeling. We present pore and fiber dimensions based on confocal microscopy and longitudinal modulus and hydraulic permeability based on confined compression. We demonstrate faithful reproduction of simple pores of 50 µm-diameter over the entire range and formation of functional microfluidic networks for mass fractions greater than 10.0 mg/ml. We present quantitative characterization of the rate and extent of cellular remodelability using human umbilical vein endothelial cells. Finally, we present a co-culture with tumor cells and discuss the implications of integrating microfluidic control within scaffolds as a tool to study spatial and temporal signaling during tumor angiogenesis and vascularization of tissueengineered constructs.

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The tocopherol transfer protein (TTP1) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analog, R) -2,5,7,8-tetramethyl-chroman-2-[9-(7-nitro-benzo[1,2,5] oxadiazol-4-ylamino)-nonyl]-chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue-shifted and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analog binds to TTP reversibly and with high affinity (Kd=8.5 ± 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the Ataxia with Vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (Kd = 71 ± 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction. Keywords affinity; fluorescence; kinetics; tocopherol; vitamin E; lipid transfer; TTP Vitamin E is a lipophilic antioxidant, initially identified as a plant-derived factor that prevented fetal resorption in diet-restricted rats (1). The term "Vitamin E" refers to a class of structurally related molecules, all of which show potent radical trapping activity in vitro (2). The ability of 1 ABBREVIATIONS NBD-TOH, (R) -2,5,7,8-tetramethyl-chroman-2-[9-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-nonyl]-chroman-6-ol; TTP, alpha tocopherol transfer protein; AVED, ataxia with vitamin E deficiency; TOH, RRR-α-tocopherol; GST, glutathione-S-transferase; IPTG, isopropyl-β-D-thiogalactopyranoside; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid, PMSF, phenylmethylsulfonyl fluoride; TRIS, tris(hydroxymethyl)aminomethane; FPLC, fast performance liquid chromatography; ABC, ATP-binding cassette transporter; Marina Blue-DHPE, Marina Blue 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; TRITC-DHPE, N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; PC, phosphatidylcholine; IgG, immunoglobulin G. § To whom correspondence should be addressed at The Division of Nutritional Sciences, 228 Savage Hall, Cornell University, Ithaca, New York, 14853. Phone: 607-255-6085; Fax: 6...

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