Differentiation of Rat Mesenchymal Stem Cells toward Osteogenic Lineage on Extracellular Matrix Protein Gradients

This report addresses the issue of optimizing extracellular matrix protein density required to support osteogenic lineage differentiation of mesenchymal stem cells (MSCs) by culturing MSCs on surface‐bound density gradients of immobilized collagen type I (COL1) and osteopontin (OPN). A chemical surface gradient is prepared by tailoring the surface chemical composition from high hydroxyl groups to aldehyde groups using a diffusion‐controlled plasma polymerization technique. Osteogenesis on the gradient surface is determined by immunofluorescence staining against Runx2 as an early marker and by staining of calcium phosphate deposits as a late stage differentiation marker. The Runx2 intensity and calcified area increase with increasing COL1 density up to a critical value corresponding to 124.2 ng cm−2, above which cell attachment and differentiation do not rise further, while this critical value for OPN is 19.0 ng cm−2. This gradient approach may facilitate the screening of an optimal biomolecule surface density on tissue‐engineered scaffolds, implants, or tissue culture ware to obtain the desired cell response, and may generate opportunities for more cost‐effective regenerative medicine. Immobilized protein‐based gradient surfaces emerge as a biological tool to facilitate the observation and changes in biological responses. By combining plasma polymerization and bioconjugation methods, extracellular matrix concentration gradients are successfully fabricated and applied for controlling stem cell behavior. This study reveals the importance of the surface density of immobilized collagen type I and osteopontin on the osteogenesis of mesenchymal stem cells.