Small Physical Cross-Linker Facilitates Hyaluronan Hydrogels

Abstract

In this study, we demonstrate that small charged molecules (NH₄⁺, GluA⁺, dHA⁺) can form physical cross-links between hyaluronan chains, facilitating polymerization reactions between synthetically introduced thiol groups (HA-DTPH). These hybrid hydrogels can be obtained under physiological conditions ideally suited for 3D cell culture systems. The type and concentration of a physical crosslinker can be adjusted to precisely tune mechanical properties as well as degradability of the desired hydrogel system. We analyze the influence of hydrogen bond formation, concentration and additional ionic interactions on the polymerization reaction of HA-DTPH hydrogels and characterize the resulting hydrogels in regard to mechanical and biocompatibility aspects.

Keywords: biocompatibility; cell encapsulation; hyaluronic acid; hydrogel; physical- and chemical cross-link; tissue engineering.

Figure A1

Swelling ratio of HA-DTPH^58%-Cl⁺ hydrogel and HA-DTPH^58%-Ox. in water and changing ionic strength and ions.

Figure A2

Exemplary graphs for the analysis of the Young’s modulus of HA-DTPH^58% without and with ionic cross-linker (1.0 equivalent ratio to remaining negative charge of HA). Young’s modulus was obtained by a linear fit of the applied strain and measured stress in the viscoelastic region of 0–5%. (a) HA-DTPH^58%-Ox (b) HA-DTPH^58%-NH₄⁺, (c) HA-DTPH^58%-GluA⁺ and (d) HA-DTPH^58%-dHA⁺.

Figure 1

Inverted tube test utilized for hydrogel synthesis with and without ionic cross-linker. (a) Chemical structure of thiol functionalized HA; (b) thiol functionalized HA (HA-DTPH^29%) with NH₄⁺, (c) HA-DTPH^29% with GluA⁺, (d) HA-DTPH^29% with dHA⁺ and (e) HA-DTPH^29% oxidized with H₂O₂.

Figure 2

Free thiol group amount within hydrogel network determined by Ellman’s assay. (a) for HA-DTPH^29%, (b) HA-DTPH^42% and (c) HA-DTPH^58%.

Figure 3

Young’s modulus of hydrogels with three thiolation degrees of HA-DTPH-Ox. and HA-DTPH-Cl⁺ hydrogels.

Figure 4

Swelling ratio of HA-DTPH^29%-Cl⁺ hydrogel and HA-DTPH^29%-Ox. In water and changing ionic strength and ions.

Figure 5

Enzymatic degradation of HA-DTPH-dHA⁺ and HA-DTPH-Ox. (a) in 100 U/mL hyaluronate lyase in PBS and (b) in 100 U/mL hyaluronidase in PBS.

Figure 6

Normal human dermal fibroblasts (NHDF) in HA-DTPH^58%-dHA⁺ hydrogel. (a) Live/Dead staining of NHDF embedded in HA-DTPH^58%-dHA⁺. Homogenous distribution of NHDF cells inside the hydrogel. Living cell (green fluorescence) and dead cell (red florescence). Image was taken as a Z-stack and max. intensity was performed via ImageJ. Scale bar = 50 µm (b) Composite images taken from NHDF embedded in HA-DTPH^58%-dHA⁺−5% RGD hydrogel. Spreading of fibroblasts (indicated with yellow arrows) was observed 24 h post-embedding inside the hydrogel. Cell nuclei are stained with DAPI. Actin is directly stained with phalloidin. Scale bar = 50 µm.