Thursday, November 8, 2007

In the US alone, roughly 1.4 million patients undergo operations requiring arterial prosthesesannually (1). Tissue engineering (TE) represents a potential means to construct functional small diametervascular grafts in situations where autologous tissue is unavailable and conventional synthetic materials fail.While initial results with many of the TE vascular grafts (TEVGs) constructed to date are very encouraging,potential for thrombosis, hyperplasia, and mechanical failure have limited their success (1). Studies suggestthat TEVG mechanical deficiencies can be attributed in large measure to smooth muscle cell (SMC) de-differentation in culture (2-9). Thus, if TEVGs are assembled with cells in a differentiated SMC phenotype,the long-term function of the neotissue will likely significantly improve. The profound effects of ECM-associated biochemical signals on cell behavior have long been recognized.However, the impact of these biochemical signals on cellular differentiation are difficult to explore in acontrolled manner, since most synthetic and natural materials adsorb a range of plasma proteins from thecell culture media. These adsorbed proteins, the levels of which are highly variable and difficult toquantitate, are major determinants of cell behavior, in addition to any molecule added by the researcher (10, 11). For controlled investigation of the effects of specific biochemical stimuli on cellular response, wetherefore need a scaffold material that is essentially “non-biofouling”. Poly(ethylene glycol) diacrylate (PEGDA )is a ”non-biofouling” material, and thus, in the absence of biochemical modification, presents a biochemical ”blank slate” to cells (12). However, the photoactivity ofPEGDA permits desired biochemical moieties to be readily conjugated into the PEG hydrogels in definedlevels (12). In the present work, we investigate the effects of a subset of biochemical stimuli believed toimpact SMC differentiation and phenotype, namely, fibronectin, laminin, fibrin, and HS. To explore theseeffects, mouse embryonic SM progenitor (10T1⁄2) cells were incorporated into PEG hydrogels to whichdefined levels of fibronectin, laminin, fibrin, or HS had been conjugated. Materials and Methods: PEGDA synthesis. PEGDA was prepared by combining 0.1 mmol/ml dry PEG (6000 Da), 0.4 mmol/mlacryloyl chloride, and 0.2 mmol/ml triethylamine in anhydrous dichloromethane and stirring under argonovernight. Synthesis of acrylate derivatized lamininand fibrin-based peptides. Laminin and fibrinogenwere be conjugated to PEG by reaction of the peptides with acryoyl-PEG-N-hydroxysuccinimide (Nektar)at a 1:1 molar ratio at pH 8.5. Synthesis of methacrylate derivatized HS. Pendant alcohol groups on HS(~16,000 Da, Sigma) will be modified with methacrylate groups to form multivinyl HS (HS–MA)macromers. Briefly, HS will be dissolved in water and reacted with an excess of methacrylic anhydride at60 C overnight at pH ~ 10. TEVG Construct Preparation and Maintenance. Hydrogels were prepared by the photopolymerization ofaqueous mixtures PEGDA. 10 mL of DMAP photoinitiator solution and 1 mM ACRL-PEG-peptide or HSMA were added per mL of aqueous mixture. The mixtures were sterile-filtered and 10T1⁄2 SM progenitorcells were suspended in each gel formulation at 1x10 cells/mL. The mixtures were pipetted between twoglass plates separated by 0.5 mm spacers and polymerized by exposure to 10 mW/cm , 365 nm UV light for 2 min. After 3 wk of culture, constructs were collected for biochemical, histological, and biomechanical