RGD ligand presentation from alginate hydrogels differentially regulates stem cell and preosteoblast phenotype.

Abstract

Musculoskeletal diseases affect more than one billion people worldwide and are one of the leading causes of long-term pain and physical disability. Traditional treatment methods are limited and bone tissue engineering has emerged as a new therapeutic alternative. Cell adhesion to extracellular matrix (ECM) ligands mediates important cellular' processes and thus, the ability to understand and control cell-ECM ligand interactions is critical for tissue engineering applications. The hypothesis driving this work is that ECM cues such as nanoscale RGD adhesion ligand presentation and substrate stiffness influence cell function and the cell response is dependent on the stage of cell commitment or differentiation. RGD peptide modified alginate hydrogels were used as model system to study the effect of ECM cues on the proliferation and differentiation of a committed cell population (MC3T3-E1 preosteoblasts) and stem cells (clonally-dervied D1 and primary human bone marrow stromal cell [hBMSC]). Whereas ECM cues influenced the proliferation and differentiation of MC3T3 preosteoblasts, they did not significantly influence the phenotype of D1 stem cells. However, when D1 cells were pre-differentiated toward the osteoblast phenotype, the cells responded to matrix cues in a similar manner as the osteoblasts. Interestingly, the response of hBMSCs to matrix cues is intermediate to that of MC3T3-E1 preosteoblasts and undifferentiated D1 cells. These results demonstrate that cellular responses to ECM cues differ depending on the cell type, and this may be related to the differentiation stage of the cell. The mechanism of the cell response-ECM cue relationship was examined using a fluorescence resonance energy transfer (FRET) technique to investigate integrin-ligand bond formation, and flow cytometry to investigate the integrin profile of preosteoblasts and stem cells. The relative bond formation increased with increasing degree of substitution (DS) of RGD modified alginates, but was not significantly altered due to changes in RGD island spacing. Flow cytometry results indicate that higher expression levels of alpha_v and alpha₅ integrins may contribute to the greater response of committed cell populations to ECM cues. These studies contribute to our understanding of the effects of ECM cues on cell function and will aid the design of biomaterials for tissue engineering applications.