Abstract: In the US alone, roughly 1.4 million patients undergo operations requiring arterial prostheses annually (1). Tissue engineering (TE) represents a potential means to construct functional small diameter vascular 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 suggest that 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 a controlled manner, since most synthetic and natural materials adsorb a range of plasma proteins from the cell culture media. These adsorbed proteins, the levels of which are highly variable and difficult to quantitate, 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, we therefore 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 of PEGDA permits desired biochemical moieties to be readily conjugated into the PEG hydrogels in defined levels (12). In the present work, we investigate the effects of a subset of biochemical stimuli believed to impact SMC differentiation and phenotype, namely, fibronectin, laminin, fibrin, and HS. To explore these effects, mouse embryonic SM progenitor (10T½) cells were incorporated into PEG hydrogels to which defined 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/ml acryloyl chloride, and 0.2 mmol/ml triethylamine in anhydrous dichloromethane and stirring under argon overnight. Synthesis of acrylate derivatized laminin- and fibrin-based peptides. Laminin and fibrinogen were 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 at 60 ^oC overnight at pH ~ 10.

TEVG Construct Preparation and Maintenance. Hydrogels were prepared by the photopolymerization of aqueous mixtures PEGDA. 10 mL of DMAP photoinitiator solution and 1 mM ACRL-PEG-peptide or HS-MA were added per mL of aqueous mixture. The mixtures were sterile-filtered and 10T½ SM progenitor cells were suspended in each gel formulation at 1x10⁶ cells/mL. The mixtures were pipetted between two glass plates separated by 0.5 mm spacers and polymerized by exposure to 10 mW/cm² , 365 nm UV light for 2 min. After 3 wk of culture, constructs were collected for biochemical, histological, and biomechanical analyses.

Biochemical Analyses. Construct cellularity (Picogreen assay), collagen (hydroxyproline assay), elastin (ninhydrin assay), and GAG (Blyscan assay) production were analyzed following overnight digestion of the constructs with 0.1 N NaOH.

Histological, Microstructural, Biochemical, and Biomechanical assays. Immunohistochemical staining for ECM proteins collagen type I and elastin, and cell differentiation indicators SM-a-actin, calponin h1,and SM-g-actin were conducted. Rectangular samples were subjected to a uniaxial strain in tension configuration until failure.

Statistical Analyses. Data sets were compared using ANOVA followed by pairwise Tukey post-hoc tests. P values less than 0.05 were considered significant.

Results/Discussion: 10T½ SM progenitor cells encapsulated in hydrogels containing HS and fibrinogen showed significantly higher levels of SM-a-actin, calponin h1, and SM-g-actin than gels containing laminin or fibronectin (Figure 1). Similarly, collagen and elastin levels were higher in the fibrinogen and HS hydrogels than in laminin and fibronectin gels (data not shown). Future investigation of the impact of systematic alterations in the biochemical composition of these hybrid scaffolds on progenitor cell differentiation should therefore yield profound insight into the dependence of cell behavior on material properties and into vasculogenesis.

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