Engineered polyelectrolyte multilayer substrates for adhesion, proliferation, and differentiation of human...

Polyelectrolyte multilayer coatings have emerged as substrates to control cellular behavior, but interactions with human multipotent mesenchymal stromal cells (MSCs) have not been studied. We looked at layer-by-layer coatings of cationic poly-L-lysine (PLL) and anionic hyaluronic acid (HA) as substrates for MSCs of placenta and adipose tissue. This system allows for modulation of thickness (number of deposition cycles), stiffness (chemical cross-linking of bulk layer), and adhesiveness (fibronectin (FN) interface). Native, as-built PLL/HA multilayer coatings were poorly adhesive for MSCs despite spectroscopy-confirmed high surface density of pre-adsorbed FN. Stratification of cross-linked PLL/HA multilayers of different stiffnesses revealed that multilayers modified with a high cross-linking regimen became efficient substrates for MSC adhesion and proliferation. MSCs on cross-linked multilayers grew to confluence. Using comparative confocal microscopy analysis of PLL/HA multilayers with physically adsorbed versus chemically coupled FN, we demonstrated that cross-linking strongly influenced FN surface distribution, leading to denser presentation of adhesion sites for cells. The covalent affixation of FN promoted focal adhesion formation and was critical to maintaining densely grown MSC cultures over weeks for their differentiation. Multilayer-bound MSCs were capable of differentiating into osteocytes and chondrocytes upon culture with induction factors. Together, cross-linked, FN-terminated PLL/HA multilayers provide a versatile platform for studies of human MSCs for biotechnological or therapeutic applications.