(267c) Combining Shear-Thinning and Temporal Covalent Crosslinking in an Injectable Hyaluronic Acid Hydrogel

justify;line-height:normal">Combining
Shear-thinning and Temporal Covalent Crosslinking in an Injectable Hyaluronic
Acid Hydrogel

justify;line-height:normal">Victoria
G. Muir, Sarah Gullbrand, Beth Ashinsky, Robert L. Mauck and Jason A. Burdick

justify;line-height:normal">Departments
of Bioengineering and Orthopaedic Surgery, University of Pennsylvania, 210
South 33rd Street, Philadelphia, PA 19104, USA

justify;line-height:normal"> 

Introduction: Hydrogels are water-swollen
networks that are used to deliver therapeutics (e.g., growth factors, cells) or
mechanical signals to musculoskeletal tissues for repair and regeneration. Injectable
hydrogels represent a class of materials that allows simple introduction into
tissues through their design (e.g., shear-thinning, gelation during
injection).  While shear-thinning properties permit easy injection, these hydrogels
often exhibit low mechanical properties; in contrast, in situ-forming
hydrogels can reach high moduli, but injection can be challenging (e.g.,
timing, extrusion from injection site during gelation). To combine
injectability and strong mechanics in the same formulation, we created an interpenetrating
network (IPN) hyaluronic acid (HA) hydrogel consisting of a shear-thinning
guest-host network (GH) and a redox-catalyzed in situ-forming covalent
network. IPN HA hydrogels were evaluated for their gelation and properties
towards biomaterials for degenerated spinal disc repair.

Materials and Methods: The guest-host network consisted
of adamantane-modified HA (AdHA, guest) and cyclodextrin-modified HA (CDHA,
host). The covalent network was formed in situ using methacrylated HA
(MeHA) with redox catalysts ammonium persulfate (APS) and tetramethyl-ethylenediamine
(TEMED). 5wt% IPN hydrogels were created by one-pot mixing of 2wt% MeHA, 3wt%
GH, 5mM APS, and 2.5mM TEMED (unless otherwise specified) in PBS (Fig. 1a). Shear oscillatory rheometry was used to assess mechanics. 5wt%
IPN HA hydrogels were injected into harvested rabbit lumbar discs ex vivo
and then in vivo into a degenerated spinal disc rabbit model.

Results and Discussion: IPN HA hydrogels exhibited
interesting properties, such as early shear-thinning and temporal covalent
crosslinking. At t = 0, 3wt% GH and 5wt% IPN were crosslinked via the GH
network, whereas the 2wt% MeHA network flowed easily (Fig. 1b and c). Onset of
gelation in the IPN was tuned by altering concentrations of redox initiator
(Fig. 1d). Furthermore, the IPN shows improved recovery of mechanical strength
after high strain compared to the MeHA network alone (Fig. 1e). IPN hydrogels
were injected into harvested rabbit lumbar discs (Fig. 1f and g). After
injection, IPN hydrogels remain localized in the cavity of the spinal disc due
to early GH crosslinking (Fig. 1g). Mechanical and histological analysis of IPN
hydrogel injections into an in vivo degenerated disc rabbit model is
ongoing.

Figure 1. (a) Schematic of
interpenetrating network chemistry. (b) Rheological analysis of hydrogels to
assess gelation kinetics. (c) Macroscopic gel behavior at t = 0 for (i) 3wt.%
GH, (ii) 2wt.% MeHA, and (iii) 5wt.% IPN. (d) Time of gelation onset for 5wt.%
IPN at varying APS and TEMED concentrations. Molar ratios of APS:TEMED
indicated. (e) Rheological analysis of storage modulus (G’) as a function of
strain (0.5 – 500%) for 2wt.% MeHA (pink) and 5wt.% IPN (blue). (f) Schematic
of (i) healthy spinal disc, (ii) degenerated, collapsed spinal disc, and (iii)
injectable hydrogels for spinal disc repair. (g) Depiction of ex vivo disc
rabbit model (i) harvested rabbit lumbar disc, (ii) injection of hydrogel into disc,
and (iii) disc cross section after hydrogel injection (blue). An ‘*’ indicates
p<0.05 between samples using Two-Way Non-Parametric ANOVA. Error bars show ±
standard deviation.

Conclusions: HA IPN hydrogels were prepared in
a one-pot mixing approach. Early mechanical properties can be tuned to achieve
clinically-relevant injectability. Furthermore, HA IPNs show promising
potential as an injectable repair strategy for degenerated spinal discs.