(393g) Improved Valvular Cell Performance in Collagen Gel-Enhanced Chitosan-GAG Scaffolds

There is great interest in a tissue-engineered approach to solving problems of heart valve replacement in children. In this study we sought to improve long-term survival and growth of valvular interstitial cells (VICs) on chitosan-based aortic valve scaffolds by increasing integrin-mediated signaling through use of collagen gels. Chitosan-based valve scaffolds were prepared from blended chitosan-collagen solution using a freeze drying method in a custom made valve mold and then surface derivatized with hyaluronan. Scaffold porosity and pore size were characterized using scanning electron microscopy; we obtained a highly porous structure with pore sizes on the aortic side larger than those on the ventricular side of the leaflets with a pore orientation favorable for trapping the cells during seeding. The dry scaffolds were neutralized with amonium hydroxide then washed with PBS followed by covalent modification with hyaluronic acid (HA) using HA-chitosan ratio 0.08. After modification, scaffolds were sterilized with 70% ethanol for 48 hrs, washed, and then equilibrated with culture medium 48 hrs and then dehydrated before seeding.Porcine VICs isolated from the aortic valves were seeded onto the dehydrated scaffolds, using either culture medium or collagen gel solution as a seeding vehicle. An initial gel solution was prepared by mixing rat tail collagen (1.11 mg/ml) with 10X DMEM in a 9:1 ratio. This initial solution was further diluted to varying levels with DMEM. VICs were cultured to 80% confluency, trypsinized, pelleted, and then resuspended in either the gel solution or medium. The suspensions were then directly pipetted onto all the scaffolds. Seeded scaffolds were incubated for 45 min at 37 ºC to allow collagen gelation. Once gelled, seeded scaffolds were overlayed with medium and cultured under static conditions for one week, followed by transfer to a custom designed bioreactor and culture under dynamic perfusion conditions. The culture medium was M199 supplemented with 15% FBS, FGF-2 and EGF (10 ng/ml each). Samples of seeded scaffolds were taken initially and weekly thereafter to evaluate cell viability and distribution.Histological evaluation of scaffolds seeded with cells suspended in medium only revealed a non-homogeneous distribution and viable cells only near the scaffold surfaces. Interior cells exhibited apoptosis within the scaffold starting during the second week of culture. In contrast, scaffolds seeded using a collagen gel showed a more uniformly distributed cell population and good cellular viability throughout the scaffold after two weeks of culture. Results indicate that the presence of a space-filling collagen gel greatly improves VIC survival and growth in chitosan-based scaffolds.