A layered agarose approach to fabricate depth-dependent inhomogeneity in chondrocyte-seeded constructs

Inspired by the depth-dependent inhomogeneity of articular cartilage, it was hypothesized that a novel layered agarose technique, using a 2% (wt/vol) top and a 3% (wt/vol) bottom layer, would create an inhomogenous tissue construct with distinct material properties in conjoined regions. The biochemical and mechanical development of these constructs was observed alongside uniform 2% and 3% constructs. Initially, uniform 3% agarose disks had the highest bulk Young's modulus (E(Y) approximately 28 kPa) of all groups. After 28 days of culture in 20% FBS-containing media, however, uniform 2% chondrocyte-seeded constructs achieved the highest Young's modulus compared to bilayered and 3% agarose disks. Though all three groups contained similar GAG content ( approximately 1.5% ww), uniform 2% agarose disks on day 28 possessed the highest collagen content ( approximately 1% ww). Unlike in either homogeneous construct type, microscopic analysis of axial strain fields in bilayered constructs in response to applied static compression revealed two mechanically disparate regions on day 0: a softer 2% layer and a stiffer 3% layer. With time in culture, this inhomogeneity became less distinct, as indicated by increased continuity in both the local displacement field and local E(Y), and depended on the level of FBS supplementation of the feed media, with lower FBS concentrations (10%) more closely maintaining the original distinction of material properties. These results shed positive light on a layered agarose technique for the production of inhomogeneous bilayered chondrocyte-seeded agarose constructs with applications for investigations of chondrocyte mechanotransduction and for possible use in the tissue engineering of inhomogeneous articular cartilage constructs.