Susan Hsiong1, Paolo Carampin2, Hyun Joon Kong2, and David J. Mooney2. (1) Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, (2) Division of Engineering and Applied Science, Harvard University, 40 Oxford Street 415, Cambridge, MA 02138
The presentation of RGD (arginine-glycine-aspartic acid) peptide sequences, common cell attachment sites present in many extracellular matrices, and the elastic modulus of the substrate presenting these peptides clearly influence the phenotype of differentiated cells. However, stem cells are increasingly used for a variety of tissue engineering applications, and it is unclear how these cells are regulated by the substrate cues. This study examined how nanoscale RGD ligand organization and substrate stiffness regulates stem cell (D1 cell line; clonally derived from mouse bone marrow) and murine preosteoblast (MC3T3-E1) proliferation using RGD modified alginate hydrogels as a model system. The pattern of RGD ligand presentation was controlled by mixing alginate polymer chains with varying RGD peptide degree of substitution with unmodified polymer chains at different ratios. The number of RGD ligands per island (2 – 40) as well as the spacing between RGD islands (36 – 121 nm) was varied while maintaining constant overall bulk density of the peptide (6.25 or 12.5 mg RGD/g alginate). The stiffness of the alginate substrate (20 – 110 kPa) was controlled by the extent of ionic cross-linking. The growth rate of MC3T3 preosteoblasts and D1 stem cells were enhanced at closer RGD island spacing, but the MC3T3 cells were twice as responsive to differences in RGD island spacing as the D1 stem cells. Increasing the elastic modulus of the gels (at a constant RGD presentation) promoted preosteoblast proliferation, whereas it had a minimal effect on stem cell proliferation. However, when D1 cells were pre-differentiated toward the osteoblast phenotype, the cells subsequently responded to RGD island spacing and gel stiffness in a similar manner as the osteoblasts. These results demonstrate that the cell response to nanoscale organization of RGD ligands and substrate stiffness is dependent on the stage of cell commitment or differentiation, and these findings will likely impact the design of biomaterials intended to direct tissue regeneration.