Designer Hydrogels for Precision Control of Oxygen Tension and Mechanical Properties

Abstract

Oxygen levels and mechanical properties provide vital cues to regulate myriad cellular functions and stem cell fate decisions. Here, we present a hybrid hydrogel system in which we can control independently oxygen levels and mechanical properties. We designed, synthesized and analyzed a hybrid hydrogel system comprised of two polymer backbones, gelatin and dextran. Both polymers were crosslinked via a laccase-mediated, oxygen consuming reaction. By specifically controlling the concentration of phenolic molecules available to react in our hydrogel, we could precisely control the time in which the hydrogel remained hypoxic (TH). We were able to achieve a range of TH from the order of minutes to greater than 10 hours. Additionally, by incorporating a secondary crosslinker, transglutaminase, mechanical properties could be adjusted in a user-defined fashion, with dynamic elastic modulus (G') values ranging from <20 Pa to >1 kPa. Importantly, oxygen levels and substrate mechanical properties could be individually tuned and decoupled in our hybrid hydrogels, while retaining the potential to study possible synergistic effects between the two parameters. By precisely controlling oxygen tension and mechanical properties, we expect that research utilizing the new hybrid hydrogels will enhance our understanding of the complex 3D cellular processes mediated by each parameter individually and may also hold clinical interest as acellular therapies.

Figure 1. Crosslinking chemistry for hybrid Gtn-FA and DexE-PT hydrogels

(A) Hypoxia inducible hydrogels are formed via laccase-mediated dimerization of phenolic moieties. Laccase catalyzes the reduction of atmospheric O₂ to water, resulting in the oxidation of phenol-containing ferulic acid (FA, ) and tyramine (TA, ) conjugated to Gtn and Dex, respectively, forming crosslinked polymer networks. (B) Additional non-conjugated phenol-containing molecules (e.g. TA, ) are added prior to the reaction, resulting in decreased cross-linking density, and thus slightly decreased stiffness. The same reaction kinetics are employed, resulting in nearly identical oxygen consumption and a decoupling of oxygen concentration and mechanical properties. (C) Secondary crosslinking via transglutaminase-mediated dimerization of primary amines ( ) present on both polymer backbones leads to increased stiffness, while not affecting oxygen consumption. By controlling the number of phenolic moieties (both conjugated and unconjugated), as well as controlling the crosslinking density by secondary crosslinking, precise control over both oxygen concentration and mechanical stiffness may be acheived.

Figure 2. Dissolved oxygen (DO) and rheological characterization for hybrid hydrogels

(A) DO levels were measured at the bottom of hydrogels (3.13 mm thick, 25 U/ml laccase) as a function of time. (B) Rheological characterization of hydrogels as measured by elastic modulus (G′) using dynamic time sweep. (C) DO and elastic modulus are coupled: as phenol concentration increases, both stiffness and time hypoxic increase.

Figure 3. Addition of non-conjugated TA enables control over TH with minimal effects on G′

(A(i) –D(i)) DO levels were measured at the bottom of hydrogels for hydrogels with addition of TA: A (Gtn-FA 3%), B (Gtn-FA 2%, DexE-PT 3.33%), C (Gtn-FA 1.5%, DexE-PT 5%), D (Gtn-FA 1%, DexE-PT 6.67%). (A(ii) – D(ii)) Rheological characterization of the same hydrogel formulations. (A(iii) – D (iii)). DO and G′ are no longer coupled and precision control over each variable is shown. Black triangles represent hybrid hydrogel TH (no TA); red triangles represent hybrid hydrogel TH with additional non-conjugated TA. ^* Indicates P<0.05

Figure 4. Dissolved oxygen (DO) and rheological characterization for hybrid hydrogels with secondary crosslinking microbial transglutaminase (mTG)

(A) DO levels were measured at the bottom of hydrogels (3.13 mm thick, 25 U/ml laccase, 0.3 U/ml mTG) as a function of time. (B) Rheological characterization of hydrogels as measured by viscoelastic modulus (G′) using dynamic time sweep and normalized to Gtn-FA 3% in order to provide a direct comparison between hydrogels. (C) DO and viscoelastic modulus remain coupled, but less strictly. ^* indicates P<0.05.

Figure 5. Vascular morphogenesis in an array of microenvironmental conditions

Gtn-FA 3% hydrogels (soft, moderate hypoxia) were amenable to network formation, which followed the well understood vascular morphogenetic process of vacuole formation, followed by lumen formation and ultimately branching and sprouting to form complex networks. Hybrid hydrogels (Gtn-FA 2%, DexE-PT 3.33%) (stiff, severe hypoxia) permitted sprouting, but not extensive network formation. Interestingly, cells seemed to aggregate and form sprouts between groups of cell aggregates without the initial stage of vascular morphogenesis (vacuole/lumen formation). Gtn-FA 3%, TA 0.054% hydrogels (soft, severe hypoxia) matched the oxygen tension in Gtn-FA 2%, DexE-PT 3.33%, but were soft. Again, cell aggregates led to sprouting, but in these softer hydrogels, more robust networks formed. Gtn-FA 3%, 0.3 U/ml mTG (stiff, moderate hypoxia) permitted the initial stages of vascular morphogenesis, but completely inhibited branching and sprouting. Vacuoles/lumen indicated by arrowheads (▶), sprouting indicated by arrows ( ). Scale bars 100 μm.