Chemically modified hyaluronic acid biomaterials for cell culture and tissue engineering

The fate and behavior of cells is strongly dependent on the cell microenvironment, and this knowledge has been applied to the design of biomaterials to influence cell growth, morphology, and differentiation. However, a dearth of research specifically focused on the effects of material hydrophobicity on cell behavior indicates that this easily controllable material property is being overlooked. The field of tissue engineering has a need for cost-efficient, scalable methods to both increase stem cell stocks and control cell behavior, and hydrophobic biomaterials may be a robust solution to these needs.

To evaluate the utility of hydrophobicity in controlling cell behavior, hyaluronic acid was modified with amines representing a wide range of hydrophobicity, resulting in twelve new materials. Both mouse embryonic stem cells (mESCs) and fibroblasts were cultured on these materials to evaluate the differences in pluripotent and differentiated cell behavior in response to hydrophobic materials. The viability of cells cultured on these materials was tested as an indicator of biocompatibility, and cell morphology and spreading area was evaluated to relate cell behavior to biomaterial hydrophobicity.

Eight of the twelve materials proved to be biocompatible, and hydrophobic materials inhibited cell spreading; fibroblasts cultured on modified HA hydrogels grew in populations of both compact cell clusters and elongated, multi-polar morphologies, and cell spreading area increased as hydrophobicity decreased. Similarly, mESC spreading area increased with decreasing hydrophobicity; mESCs grown on the least hydrophobic HA hydrogels also multi-polar spreading, while mESCs cultured on the more hydrophobic materials grew exclusively in compact cell clusters.

As the morphology of cells is often indicative of cell fate, and as hydrophobic materials tended to inhibit cell spreading, we expected that mESCs cultured on hydrophobic materials would maintain pluripotency. To this end, hybrid scaffolds composed of modified HA and gelatin were developed as a platform for stem cell pluripotency maintenance. The mESCs seeded into these scaffolds had a higher expression of the pluripotency marker SSEA-1 compared to control mESCs grown in complete medium after 24 hours, indicating that hydrophobicity is an important material property to consider in stem cell culture.