Independent control of molecular weight, concentration, and stiffness of hyaluronic acid hydrogels

Abstract Hyaluronic acid (HA) hydrogels have been used for a multitude of applications, perhaps most notably for tissue engineering and regenerative medicine, owing to the versatility of the polymer and its tunable nature. Various groups have investigated the impact of hydrogel parameters (e.g. molecular weight, concentration, stiffness, etc) in vitro and in vivo to achieve desired material performance characteristics. A limitation in the literature to date has been that altering one hydrogel parameter (a ‘manipulated variable’) to achieve a given hydrogel characteristic (a ‘controlled variable’) changes two variables at a time (e.g. altering molecular weight and/or concentration to investigate cell response to stiffness). Therefore, if cell responses differ, it may be possible that more than one variable caused the changes in observed responses. In the current study, we leveraged thiol-ene click chemistry with a crosslinker to develop a method that minimizes material performance changes and permitted multiple material properties to be independently held constant to evaluate a single variable at a time. Independent control was accomplished by tuning the concentration of crosslinker to achieve an effectively constant stiffness for different HA hydrogel molecular weights and polymer concentrations. Specific formulations were thereby identified that enabled the molecular weight (76–1550 kDa), concentration (2%–10%), or stiffness (∼1–350 kPa) to be varied while the other two were held constant, a key technical achievement. The response of rat mesenchymal stem cells to varying molecular weight, concentration, and stiffness demonstrated consistent upregulation of osteocalcin gene expression. The methodology presented to achieve independent control of hydrogel parameters may potentially be adopted by others for alternative hydrogel polymers, cell types, or cell culture medium compositions to minimize confounding variables in experimental hydrogel designs.