(668d) Mapping of Collagen and Hyaluronic Acid Hydrogel Properties to Functional Responses

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 and migration. Despite the widespread use of collagen and HA (ColHA) hydrogels in tissue engineering, the vast design space has not been mapped yet due to the large number of fabrication parameters that could be investigated. This limitation stems from experimental difficulties, as traditional methods of hydrogel characterization include studying all possible fabrication parameter combinations and require three replicates. This traditional approach is labor intensive, time consuming, and expensive due to the large amount of material needed. In our study, we develop a mathematical model based on Latin hypercube experimental design and the Bayesian inference method. The model utilizes empirical data collected to predict the effect of the fabrication parameters (collagen polymerization temperatures, HA concentrations and MW simultaneously across large ranges) on the hydrogel properties (collagen and HA retention, microstructure, mechanical properties) and functional responses (transport and cell behavior) of the hydrogel.

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 in vivo tissues and processes. Numerous matrix polymerization temperatures (15, 20, 25, and 37 °C) were investigated. The collagen incorporation into the ColHA gels after 14 days is nearly 100% for all matrices tested. The HA is released from the gel over time. The HA retention in the gel is related to polymerization temperatures of the matrices, and HA MW. Higher polymerization temperatures and higher MW HA result in higher HA retention. Microstructure of ColHA hydrogels is governed by polymerization temperature. Polymerization temperature and pore diameter are inversely related, as assessed by algorithm developed by Antoine at al 2014.¹ Smaller pore sizes of hydrogels polymerized at higher temperatures help explain increase in HA equilibrium concentration in hydrogels polymerized at higher temperatures. ColHA gels polymerized at 25 °C resulted in gels with the highest storage modulus (G’), while gels polymerized at 15 °C resulted in lowest G’. HA did not have a significant impact on mechanical properties of the hydrogels. A Transwell mass recovery assay, a technique which investigates molecular transport through biological barrier, was performed to assess how different matrix properties alter diffusion profiles. Results demonstrate that transport of bovine immunoglobulin G (IgG) is related to the pore sizes of the matrix, as hydrogels with larger pore sizes experience faster recovery compared to hydrogels with smaller pore sizes. Furthermore, inclusion of HA (a viscous polymer) decreases the recovery rate of IgG.

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. The mathematical model developed helped to characterize large design space, and map the fabrication parameters to material properties, and functional properties (such as transport). The hydrogels can be tailored to study therapeutic diffusion and recovery.

¹Antoine, E. E., Vlachos, P. P. & Rylander, M. N. Tunable collagen I hydrogels for engineered physiological tissue micro-environments. PLoS ONE 10, 1–18 (2015).