(540f) Strain-Stiffening Hydrogel with Tunable Water Content

Polymer hydrogels are three-dimensional networks composed of crosslinked polymer chains that are capable of retaining a large amount of water within their structures. Hydrogels show several physiochemical features similar to biological tissues (owing to their water abundance, intrinsic flexibility, and microstructures) and hence superior to traditional biomaterials such a plastics or ceramics when used in biomedical applications.¹ However, conventional hydrogels possess two significant drawbacks: (i) the initial stiffness of the gels is fixed and cannot be modified post-synthesis, and (ii) the gels usually weaken when subjected to repeated strain and over time during culture with cells. Also, the high water content of hydrogels generally limits their stiffness, as high swelling is associated with large mesh sizes. We sought to overcome these limitations by combining an ultra-hydrophilic monomer phosphorylcholine (PC) with a co-monomer HEMA-LA that could undergo strain stiffening to create a highly hydrophilic, yet mechanically robust strain-stiffening hydrogel network.

To create this hydrogel, we took inspiration from proteins. Several biological soft tissues such as skin, lung, blood vessels, etc., can become stiffer upon increasing stress as a mechanism to maintain the integrity of the tissues and homeostasis.² Tissues do this by incorporating proteins into the extracellular matrix, which can polymerize or form networks with other proteins whilst they unfold under external stress. Examples of these mechano-sensitive proteins include actin cytoskeletal filaments, which polymerize under cell strains³, fibrin gels that form during blood clotting⁴, and fibronectin that, under deformation, reveals a “cryptic site” that can bind additional fibronectin and polymerize.⁵ These biological systems have accelerated the research to design strain sensitive synthetic materials with strain-induced strengthening.⁶ However, such hydrogel systems are limited, and realizing a generic approach to install mechano-sensitive stiffening in hydrogels would significantly broaden their application arena.

Most of the innovations on mechano-responsive hydrogels utilize mechanophores that are activated upon mechanical stress.⁷ This approach provides on-demand strengthening with high spatial control. However, the mechanical energy required for the activation is often too high for biomedical applications. On the other hand, few sophisticated fabrication approaches have demonstrated temporary gel stiffening due to the inter-chain association but have not achieved permanent crosslinking/ strengthening.⁸ Therefore, a simple and scalable approach having the potential for strain-stiffening hydrogels with ‘low energy’ mechanical activation is highly desired.

In this conference presentation, we will describe a simple strategy to construct a hydrogel with strain-sensitive stiffening properties by utilizing the cryptic crosslinking domains. These domains consisted of a thiol bearing monomer buried within the poly(ethylene glycol) (PEG)-acrylate/phosphocholine (PC)-methacrylate backbone. The PEG chains effectively shield the thiol-based crosslinkers. However, upon the mechanical activation, the PEG groups would un-shield the crosslinkers and effectively promote the interchain crosslinking, thereby stiffen the hydrogel network. The water content in the hydrogel matrix could be tuned by varying the content of the phosphocholine (Figure -1). This strategy would expand the applications of hydrogels by providing a brand-new type of latency and strain-induced crosslinking.

Methods.

Hydrogel synthesis. The hydrogel polymer network was synthesized by copolymerizing the 2-hydroxyethyl acrylate (HEA), 2-hydroxyethyl methacrylate-lipoic acid (HEMA-LA),2-methacryloyloxyethyl phosphorylcholine (PC) and poly(ethylene glycol) dimethacrylate (PEGDMA) in DMSO solvent and by using AIBN as a radical initiator. The resultant gel under-went dialysis for 7 days with water replaced at least four times a day. Finally, the solvent exchanged gel was subjected to freeze-drying. The dried polymer network was then placed in water for 2 days to obtain fully swelled hydrogel. A series of hydrogels were synthesized by varying the amount of phosphorylcholine in the polymer network (0- 20 mol %). The hydrogels with PEG shielding groups were synthesized by replacing the HEA monomer with a PEG containing monomer (PEGMA).

Hydrogel characterization. The equilibrium water content in the hydrogel was determined by taking dry-weight and the swollen-weight of the sample. The mesh-size of the hydrogel was determined according to the modified Flory theory.⁹ The hydrophilicity was also monitored by contact angle measurements in captive bubble configuration using a home-made setup. The strain-induced crosslinking studies are performed on a parallel plate rheometer (AR 2000, TA Instruments). The cylindrical hydrogel was placed between the plates, and the cyclic compression/tension tests were conducted with a rate of 20 µm/s within a 0-25 % strain range.

Results.

We synthesized 5 sets of hydrogels consisting of 20 mol% of HEMA-LA and varying amounts of HEA (67 – 47 mol%) and PC (0 – 20 mol%) in the DMSO solvent (Figure 2a). The gel was put through dialysis for solvent exchange with water for 7 days. The solvent exchange was studied by proton NMR spectroscopy, and the DMSO in the hydrogel immersed water was monitored (Figure 2b). The resultant hydrogel was subjected to freeze-drying to get the dry polymer network. The hydrogel state was recovered by immersing the polymer network in water for 2 days. The equilibrium water content was determined by accurately weighing the dry-weight and the swollen-weight. Interestingly, the equilibrium water content in the hydrogel showed a linear relationship with the phosphocholine (PC) content (Figure 2c). The hydrogel with no PC showed insignificant water content; however, the hydrogel with 20 mol% of PC showed an equilibrium water content as high as 60 %. In addition, we tested the swelling in 1X PBS (pH 7.4) for future biological application. The results were similar, but the water intake was slightly lower than that of pure water (Figure 2d).

In order to demonstrate the strain-sensitive crosslinking, the as-synthesized hydrogel (HEA was replaced with PEG shielding groups) was subjected to the chemical reduction. This step activates the lipoic acid (LA) residues and generates the thiol groups, which serve as latent crosslinking sites (Figure 1b). These sites are buried within the steric shielding of the PEG units; hence, the interchain crosslinking could only occur if the free thiol groups are accessible to each other. The compressive force provides a convenient means to displace the PEG chains so that the thiol groups could react and generate interchain crosslinks. We studied the process of interchain crosslinking as a function of time under the compressive strain (Figure 3, red curve). The hydrogel showed a significant enhancement of the stiffness over time (almost 5 times compared to the original stiffness). However, a control sample that was not subjected to any compressive strain showed on a change of about 3 times the initial stiffness (Figure 3, black curve). This clearly suggests the force activated crosslinking in the hydrogel.

In summary, we have demonstrated an approach to install mechano-sensitive stiffening in hydrogel network. The method utilized reversible thiol crosslinker chemistry on a PEG appended acrylate backbone network. In addition, the phosphocholine motifs proved to be a useful parameter to tune the water content in the hydrogel.

Future Work.

Our preliminary results have shown the approach to synthesize mechano-responsive stiffening hydrogels. Next, we will determine the contact angle of the hydrogel as a function of PC concentration. The amount of residual free thiol groups will be quantified as a function of crosslinking time to monitor the process in detail. We will also study the effect of the direction of strain on the crosslinking process. Finally, we plan to grow cells on these hydrogels to quantify the effects of cell-generated forces on hydrogel stiffening.

References.

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