Fabrication of micro-porous hyaluronic acid hydrogels through salt leaching

Development of physiologically relevant culture platforms for studying cell behavior has been of particular interest in tissue engineering as well as in development of in vitro disease models. Hyaluronic acid is a glycosaminoglycan found in the extracellular matrix, derivatives of which are commonly used to create three-dimensional biomimetic hydrogel scaffolds for the study of cell behavior in vitro. Herein, we report the fabrication of micro-porous hyaluronic acid hydrogels using a simple salt leaching technique. Porogens, specifically, salt particles of different sizes (i.e., < 63 mu m, 63-106 mu m, 106-250 mu m in diameter), were utilized to obtain a range of porous structures. The microstructure of the hydrogels was characterized through scanning electron microscopy, and rheological and swelling studies were performed to further evaluate morphological and mechanical properties. Analysis of these studies indicated that an increase in pore area corresponded to a larger porogen size, whereas porogen size had no significant effect on storage modulus or water absorption. However, porous hydrogels exhibited a greater degree of swelling and reduced storage modulus compared to nonporous hydrogels. In particular, the largest particle size of 106-250 mu m resulted in a swelling ratio of 44.6 +/- 2.28 and storage modulus of 54 +/- 4.97 Pa versus a swelling ratio of 24.3 +/- 2.30 and storage modulus of 402 +/- 209 Pa noted for nonporous hyaluronic acid hydrogels, respectively. No significant difference in degradation of hydrogels was observed at later time points; however, hydrogels with the largest pore area degraded to a greater extent initially. Assessment of cellular compatibility showed no evidence of cytotoxicity for all hydrogel conditions tested. Such a system, incorporating micron-level porosity, could provide a useful tool for studying cell-matrix interactions in a physiologically relevant setting.