Effect of Initial Cell Seeding Density on Early Osteogenic Signal Expression of Rat Bone Marrow Stromal Cells Cultured on Cross-Linked Poly(propylene fumarate) Disks

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Abstract
The intercellular signaling mechanisms among a transplanted cell population are largely determined by the cell population itself as well as the surrounding environment. Changes in cell-to-cell paracrine signaling distance can be obtained by altering cell density, and signal expression of growth factors can be enhanced by auto/paracrine signal transduction. To examine these relationships, we investigated the effect of cell seeding density on viability, proliferation, differentiation, and the endogenous osteogenic signal expression among rat bone marrow stromal cells (BMSCs) cultured on a 2D disk. Rat BMSCs were isolated from rats and then cultured for 8 days on biodegradable poly(propylene fumarate) disks with three different seeding densities (0.06, 0.15, and 0.30 million cells/disk). At days 1, 4, and 8, viability by live/dead fluorescent staining, DNA amount, osteogenic differentiation by alkaline phosphatase and osteocalcin mRNA expression, calcium deposition, and osteogenic growth factor mRNA expression were assayed. Osteogenic signal expression was evaluated using quantitative reverse transcriptase-polymerase chain reaction, and signals of interest include bone morphogenetic protein-2, transforming growth factor-β1, fibroblast growth factor-2, and platelet-derived growth factor-A. The results from this study demonstrate that rat BMSCs were viable over 8 days without being affected by cell density and that both cell proliferation rate and early osteogenic differentiation were stimulated by lower cell seeding density. Most importantly, this study has demonstrated for the first time that the temporal gene expression profiles of endogenous growth factors can be controlled by altering the initial cell seeding density on poly(propylene fumarate) disks. Therefore, our results suggest that changes in the paracrine signal distance by altering cell seeding density may be a useful strategy to optimize the cell-biomaterial construct microenvironments to enhance the osteogenic signal expression.