Synthesis And Characterization of Leucine Zipper Hydrogel For Tissue Regeneration

Tissue engineering is based on three major factors: cells, biological signals, and scaffold. Biomaterials are currently being developed for biological activity, and the essential property required for a functional scaffold is cell attachment and cell viability for tissue engineering applications. Scaffolds are also required to deliver specific biological signals at the specific time. The Leucine Zipper (LZ) is a secondary α-helical coiled-coil structure found in several proteins. The LZ hydrogel is a novel biomaterial and is formed by the inherent self-assembly property of the LZ domain in the chimeric protein. However, the current LZ hydrogel is not stable enough to function as a scaffold for a desired period of time. Furthermore, the LZ hydrogel is not yet developed and explored as a scaffold. In this study, we developed a stable 3-D LZ hydrogel by using a dehydrothermal crosslinking method. The LZ hydrogel is porous. By altering the weight percentage of the hydrogel, the pore size and the mechanical properties can be changed. The LZ hydrogel is also tunable with respect to its functionality by incorporating different functional domains. We evaluated several different functional motifs after incorporated them into the LZ hydrogel, including RGDS, DMP-1 derived calcium-binding domains (ESQES and QESQSEQDS) and a heparin-binding domain with a MMP-2 cleavage site. The LZ hydrogel by itself can provide good cell affinity for 3-D cell culture and did not trigger foreign body reaction when implanted subcutaneous in a wild type mouse. The RGDS incorporated LZ hydrogel can provide an even better cell culture environment by facilitating the cell attachment, proliferation and migration. The LZ hydrogel incorporated with DMP-1 derived calcium-binding domains showed the ability of accumulating calcium ions and further triggered biomineralization in vivo. The incorporation of heparin-binding domain with MMP-2 cleavage site facilitated tethering of heparin-binding growth factors (VEGF, TGF-β1 and BMP-2) and controlled release of active growth factor in vitro and in vivo. Overall, we have shown that LZ, a self-assembling peptide can be utilized for the synthesis of a defined matrix for tissue regeneration. The incorporated functional domains can also provide the hydrogel with tunable properties for varied applications. This is the first report on the development of a stable LZ hydrogel with tunable properties.