Polyacrylamide hydrogels were prepared with variable stiffness within a range of effective surface Young's modulus values from 5.5 kPa to 152 kPa measured in the hydrated state using atomic force microscopy (AFM). The gel surface was modified with either collagen or plasma polymer coatings containing amino, carboxyl or phosphate moieties. Analysis of the surface chemistry using X-ray photoelectron spectroscopy and AFM indentation showed that the coated gels present very different surface chemistries while maintaining the range of stiffness. The density of human mesenchymal stem cells (hMSC) adhered to the materials was found to depend on the surface chemistry, with the highest cell densities achieved for collagen coated gels. The spread of each cell was shown to be greater for the stiffer surfaces independent of surface chemistry. To assess the differentiation of the hMSCs, antibody staining was carried out using markers for osteogenic (Runx2), myogenic (MyoD1) and neurogenic (β-III tubulin) cell types which revealed a dependence of marker protein expression upon both surface stiffness and chemistry. The expression of the osteogenic Runx2 marker was maximal for cells cultured on gels of 41 kPa stiffness when modified with the phosphate plasma polymer. Myogenic MyoD1 expression was maximal on the carboxyl coated gels of intermediate stiffness (10 kPa to 17 kPa). Neurogenic differentiation indicated by β-III tubulin expression was seen to be greatest on the carboxyl surfaces and for the lowest surface stiffness substrates. Using soluble factors in the medium to induce osteogenic behaviour resulted in the formation of bone nodules and matrix calcification for gel stiffness values higher than 10 kPa, especially on amino-functionalized coatings but not for collagen coated gels. The results indicate that control over differentiation fate of hMSCs can be exerted using not only surface stiffness, a result previously widely reported, but also surface chemistry working in tandem with the influence of compliance. This has great significance in developing stem cell therapies when synthetic surfaces are used as scaffolds, delivery vehicles or culture ware.
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