(43a) Proteolytic Degradation of Hyaluronic Acid Hydrogels for Controlled Growth Factor Delivery

Growth factors play an important role in activating and regulating various cellular functions. One common approach to control growth factor delivery is through the manipulation of diffusion through hydrogels. Release rates can be further controlled by introducing degradation, such as through hydrolysis; however, hydrolysis can be limited as a means to control release kinetics as growth factor release profiles due to hydrolysis rates are somewhat limited. As an alternative, we designed hydrogels with proteolytically controlled molecule release. Specifically, maleimide-modified hyaluronic acid (MaHA) was synthesized and used as a platform for incorporation of proteolytically sensitive crosslinks and cell-adhesive peptides via a Michael Type addition reaction. Maleimide functionalization (f) was varied between 10 to 40%f based on the maleimide to HA ratio during coupling and was assessed with ¹H NMR. For gelation, MaHA was dissolved in phosphate buffered saline (PBS) with or without growth factors. Cell adhesive and matrix metalloprotease (MMP) sensitive peptides were added and reacted according to a Michael Type addition reaction between thiols (via cysteine groups) and maleimides, allowing for protease-mediated hydrogel degradation.

The compressive and rheological properties of MaHA hydrogels were characterized as a function of hydrogel polymer content (1-5 wt%) and functionalization (10-40%f). As expected, the compressive modulus increased with both polymer content and functionalization. At 40% functionalized MaHA, the compressive modulus increased from ~2 to 21 kPa for hydrogels with 1 and 5 wt% MaHA, respectively. Rheology indicated reaction rate was dependent on hydrogel formulation, where the crosslinking reaction occurred faster at higher polymer contents and functionalization. Time to 90% of the plateau modulus ranged between approximately 3 (40%f, 5 wt% MaHA) and 80 (10%f, 3wt% MaHA) minutes. MaHA hydrogel degradation and growth factor release (bone morphogenic protein; BMP2) was performed in 0, 1, 2, and 10 U/ml collagenase (non-specific MMP degradation) at 37°C. Hydrogel degradation studies indicate that the hydrogels undergo rapid degradation in the presence of MMPs (collagenase) and little degradation without MMPs. Furthermore, growth factor release occurred coincident with hydrogel mass loss, with little release in the absence of MMPs. These results indicate that release from this class of hydrogels is primarily protease-mediated, rather than through hydrolysis or diffusion and can be engineered with a range of release profiles.