Developing an Empirical Model for Designing Tunable Collagen and Hyaluronic Acid Blended Hydrogels

Collagen I and hyaluronic acid (HA) are commonly utilized to form hydrogels that mimic the extracellular matrix for tissue engineering applications. Scaffold microstructure, component incorporation, and modulus govern molecular transport and provide biochemical and mechanical signals for cellular proliferation, differentiation, and migration. Despite their widespread use in tissue engineering, the physical and mechanical properties of these materials are variable across studies utilizing similar conditions. In our study, we developed an empirical model toward the formation of tunable, well-defined, and robust collagen-HA blended hydrogels (ColHA) for a wide range of tissue engineering applications.<br/><br/>Statistical modeling was utilized to fully characterize the design space of the collagen and HA blended hydrogels. In particular, we probed the contribution of parameters, such as HA concentration, HA molecular weight, and the polymerization temperature of collagen, to modulate materials properties, including scaffold modulus, microstructure, and functional assays such as molecular transport through the gels. Hydrogels were formulated with 4 mg/mL of collagen, and physiologically relevant HA concentrations (0, 0.5, 1, and 2 mg/mL) and molecular weights (50, 500, and 1500 kDa) were chosen to fully represent various <i>in vivo </i>tissues and processes. It is known that the entanglement point, or the concentration at which polymer chains form entangled networks, of 1500 kDa HA is around 1 mg/mL.¹ Thus, the molecular weights and concentrations chosen reflect HA behavior below, at, and above the entanglement point. Numerous matrix polymerization temperatures (15, 20, 25, and 37 °C) were investigated to capture the full range of fibril formation capabilities of collagen.<br/><br/>Robust fabrication protocols were developed and validated by five individuals reproducing the matrix and resulted in uniform component incorporation and microstructure. The collagen incorporation into the ColHA gels after 14 days is nearly 100%, and thus independent of polymerization temperature and HA presence in the gels. The HA incorporation appears to be directly related to the entanglement point of HA. The gels consisting of HA above entanglement point appear to have slower diffusion of HA out of the ColHA gels compared to those gels containing HA below the entanglement point. Polymerization temperature appears to have an effect on gel swelling as the percent mass change is smallest for gels polymerized at 37 °C and largest for gels polymerized at 15 and 20 °C. The changes in microstructure that arise from varying polymerization temperatures result in a range of mechanical properties. ColHA gels polymerized at 25 °C resulted in gels with the highest complex modulus (G*), which was three times higher than those of gels polymerized at 37 °C. A Transwell mass recovery assay was performed to assess how different matrix properties alter diffusion profiles. The Transwell assay highlighted the importance of electrostatic charge and viscous matrix effects for mass transport efficiency of various molecules through the matrices. Overall, the results of this study demonstrate that ColHA gels span a large design space that can be tailored for tissue engineering applications with specific mechanical properties, fibril microstructure, equilibrium HA concentration, and macromolecule transport profiles.<br/><br/><br/>¹Morris, E. R., Rees, D. A., Welsh, E. J. Conformation and Dynamic Interactions in Hyaluronate Solutions. <i>J. Mol. Biol.</i> (1980) <b>138</b>, 383-400