Effect of Seeding Osteoprogenitor Cells as Dense Clusters on Cell Growth and Differentiation

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Abstract
One approach for forming tissue equivalents involves seeding of cells into porous scaffolds followed by culture in vitro. Within this paradigm, the strategy by which cells are initially seeded may dictate the ultimate properties of the tissue equivalent. In particular, low cell densities may suffer from poor intercellular communication, whereas high densities may result in an unfavorable microenvironment due to transport limitations. A third alternative is to seed cells as dense clusters, which might benefit from intercellular contact without the high nutrient demand. To test this approach, planar substrates were seeded with 104 osteoprogenitor marrow stromal cells either as a diffuse subconfluent dispersion (2.6 × 103 cells/cm2) or as a single dense cluster (8 × 104 cells/cm2). In this study, the densely clustered cells demonstrated significantly diminished cell growth and collagen synthesis. However, a significantly higher level of alkaline phosphatase activity—a measure of bone-forming potential—and moderately more mineralization were observed with these dense cultures. These findings show that clustering can enhance the differentiation phase while diminishing the proliferating phase of these diploid cells without requiring large cell numbers. Thus, this seeding strategy may improve the quality of engineered tissues.