Cellular Utilization Determines Viability and Matrix Distribution Profiles in Chondrocyte-Seeded Alginate Constructs

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Abstract
The long-term success of any cellular construct used for cartilage tissue engineering is dependent on the maintenance of cell viability throughout the construct thickness. Furthermore, the cells must continue to be metabolically active in order to synthesize a mechanically functional extracellular matrix (ECM). In the present study, a live–dead staining technique and systematic profiling procedure enabled the spatial and temporal distribution of chondrocyte viability to be characterized within 4-mm-thick alginate scaffolds. ECM distribution after 14 days of culture is described both biochemically and histologically and the mechanical functionality of the constructs was assessed by an unconfined compression test. Parameters investigated included alginate permeability, cell-seeding density, and volume of culture medium. Nonhomogeneity of cell and matrix distribution was evident, with greater densities of both parameters in the periphery of the constructs. The culture time preceding central viability loss was inversely related to cell density but relatively independent of scaffold density. However, homogeneity could be attained with increasing medium volume, as evidenced with cell and matrix distribution for cultures in 6.4 mL of medium per 106 cells. Moreover, the mechanical properties of the construct were enhanced by culture in increasing volumes of medium. This work indicates that cellular utilization determines the nonhomogeneous nature of cartilage formation in three-dimensional constructs and presents a guide to nonlimiting medium volumes for static culture conditions.