Introduction: In recent years, tough hydrogels have become a topic of interest, as their resilience toward mechanical failure perpetuates use in load bearing applications and long-term implantation (e.g., cartilage, intervertebral disc)[1]. However, double networks (DN) with these desirable mechanics that are also injectable and amenable to the encapsulation of viable cells have not been developed, hampering translation in vivo. Here, these features are demonstrated through a combination of extensible covalent networks and supramolecular guest-host networks.
Materials and Methods: Hyaluronic acid (HA) hydrogels were formed (Fig 1A) from methacrylated HA (MeHA, 100% modified) by addition reaction with dithiothreitol (DTT; ratio thiol/methacrylate = 0.2; pH 8 overnight). Secondary networks of a guest-host hydrogel of adamantane and cyclodextrin HA (Ad-HA & CD-HA, 30% modified) were included by physical interpenetration (GH DN) or with covalent crosslinking between the two networks (MethGH DN; 20% methacrylated Ad-HA and CD-HA). Mechanical analysis was performed in compression (TA Q800). Cell viability was examined at in networks composed of acrylated HA (AHA)[2] and the associated GH DN.
Results and Discussion: Supramolecular double networks were investigated for their ability to impart increased moduli and failure stress into extensible covalent networks. Compressive analysis to failure (Fig 1B) demonstrated increased failure stress for GH DN (335±30 kPa), compared to MeHA alone (163±30 kPa). Compressive moduli were increased by DN formation and greatest for MethGH DN (Fig 1C). Similar results were attained at increased DTT concentration, with moduli of MethGH DN (226±23 kPa) demonstrated, exceeding MeHA (86±4.5 kPa). Loading to 90% strain (Fig 2A) demonstrated different modes of failure: brittle (MeHA), ductile (GH DN), and recovery (MethGH DN). Repeated compression to >90% of failure strain (80%, Fig 2B) was used to assess damage from repeated loading which was apparent in MeHA, attenuated in GH DN, and absent in MethGH DN. In these same tests, initial moduli were immediately recovered in MethGH DN, but not other networks. These results indicate internal, irrecoverable damage for MeHA and GH DN, whereas MethGH DN exhibited rapid internal self-healing and absence of the Mullins effect characteristic of other DN gels[3]. While Michael addition with MeHA requires long reaction times (pH 8, overnight), adaptation to AHA enabled rapid crosslinking to achieve high cell viability (>90%, Fig 2C), and is amenable to crosslinking at physiological pH (i.e., after injection in vivo).
Conclusions: Supramolecular secondary networks were used to alter hydrogel properties. Covalent coupling between the networks (MethGH DN) improved mechanics over interpenetration alone (GH DN), likely due to improved stress transfer. Adaptation to acrylated materials enabled transfer of these properties to networks whose crosslinking occurs under cytocompatible conditions.
Financial support provided by a Predoctoral Fellowship (C.B.R.) and Established Investigator Award (J.A.B.) from the American Heart Association.
References:
[1] Costa AMS & Mano JF. European Polymer Journal. 2015.
[2] Marklein RA et al. Soft Mater. 2012.
[3] Webber RE, et al. Macromolecules. 2007.