Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels

With advanced understanding of how manipulations in material chemistry and structure influence cellular interactions, material control over cellular behavior (e.g., spreading) is becoming increasingly possible. In this example, we developed a novel process that utilizes different crosslinking mechanisms to provide gel environments that are either permissive or inhibitory to cellular spreading. To accomplish this, a multi-acrylated macromer (i.e., acrylated hyaluronic acid) was first crosslinked with an addition reaction using a matrix metalloprotease (MMP) cleavable peptide containing thiol groups. When an adhesive peptide was also coupled to the network, this environment permitted the spreading of encapsulated human mesenchymal stem cells (hMSCs), whereas control systems did not. If all acrylates were not consumed during the initial crosslinking step, a photoinitiated radical polymerization could be used to crosslink the remaining acrylates and inhibit cellular spreading with the production of covalent barriers. Variations in the ratio of the two crosslink types in individual constructs controlled the degradation and mechanical properties of the hydrogels, as well as the degree of spreading of encapsulated cells. Cell spreading was further controlled spatially with the use of photomasks. Overall, this new technology is an exciting and potentially valuable tool, both to provide new insights into the relationships between gel structure and cell behavior, and for eventual tissue-engineering applications where spatial control over cells is desired.