Development of Mesoporous Silica Nanoparticle-Based Films with Tunable Arginine-Glycine-Aspartate Peptide Global Density and Clustering Levels to Study Stem Cell Adhesion and Differentiation

Stem cell adhesionis mediated via the binding of integrin receptorsto adhesion motifs present in the extracellular matrix (ECM). Thespatial organization of adhesion ligands plays an important role instem cell integrin-mediated adhesion. In this study, we developeda series of biointerfaces using arginine-glycine-aspartate(RGD)-functionalized mesoporous silica nanoparticles (MSN-RGD) tostudy the effect of RGD adhesion ligand global density (ligand coverageover the surface), spacing, and RGD clustering levels on stem celladhesion and differentiation. To prepare the biointerface, MSNs werechemically functionalized with RGD peptides via an antifouling poly(ethyleneglycol) (PEG) linker. The RGD surface functionalization ratio couldbe controlled to create MSNs with high and low RGD ligand clusteringlevels. MSN films with varying RGD global densities could be createdby blending different ratios of MSN-RGD and non-RGD-functionalizedMSNs together. A computational simulation study was performed to analyzenanoparticle distribution and RGD spacing on the resulting surfacesto determine experimental conditions. Enhanced cell adhesion and spreadingwere observed when RGD global density increased from 1.06 to 5.32nmol cm(-2) using highly clustered RGD-MSN-based films.Higher RGD ligand clustering levels led to larger cell spreading andincreased formation of focal adhesions. Moreover, a higher RGD ligandclustering level promoted the expression of alkaline phosphatase inhMSCs. Overall, these findings indicate that both RGD global densityand clustering levels are crucial variables in regulating stem cellbehaviors. This study provides important information about ligand-integrininteractions, which could be implemented into biomaterial design toachieve optimal performance of adhesive functional peptides.