Dynamic Composite Hydrogel with Programmable Swelling-Shrinking for 4D Bioprinting

Introduction: : The field of three-dimensional (3D) bioprinting is limited by static hydrogels that fail to replicate the dynamic nature of biological systems. To fill this gap, hydrogels with programmable swelling-shrinking properties have been investigated for applications such as the absorption of blood and wound exudates, nutrients and metabolite transfer, on-demand drug release, and complex shape transformation in 4D printing. However, conventional hydrogels swell less than 20% of their original volume before mechanical failure and lack reversible shrinking capacity. To overcome these challenges, we engineered a biocompatible, stimuli-responsive, high-swelling composite hydrogel by combining gelatin methacrylate (GelMA), sodium polyacrylate (SPA), and poly(ethylene glycol) diacrylate (PEGDA). This high-swelling hydrogel, hereafter referred to as SwellMA, exhibited a 100-fold increase in water uptake while offering rheologic properties suitable for 3D printing. SwellMA also demonstrated controlled, cyclic swelling-shrinking in response to osmolarity changes, along with enhanced cell adhesion, proliferation, and biocompatibility compared to GelMA and SPA. These findings establish SwellMA’s as a promising platform for advanced 4D bioprinting and a broad range of biomedical applications requiring precise and dynamic control over hydrogel swelling and shrinking.

Materials and

Methods: : We synthesized SwellMA by adding GelMA 10% w/v to a solution with a molar ratio of 37% sodium acrylate and 67% acrylic acid. The solution also included a crosslinker PEGDA 0.25% w/v, and the photoinitiators Omnirad 2100 1% w/v and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) 0.5% w/v. The solution was mixed homogenously and crosslinked under ultraviolet (UV) light after casting into molds or 3D-bioprinted (Fig. 1A). Swelling characterization was done through different concentrations of the crosslinker PEGDA, measuring surface area change with swelling, and the fold change of water weight. Cyclic swelling-shrinking properties were measured through repeated cycles of swelling in phosphate-buffer saline (PBS) and shrinking in 100 mM calcium chloride. Rheology properties were measured on swelled samples taking a frequency sweep at 1 % strain and strain sweep at 1 Hz to measure storage modulus(G’) and loss modulus (G’’). Cell adhesion and viability were analyzed seeding 1 x 105 rat bone marrow-derived mesenchymal stromal cells (MSC) on swelled SwellMA gels then conducting fluorescent imaging and flow cytometry. Biodegradation was measured after 14 days of treatment with collagenase type II using concentrations found in physiological wound healing. Printing fidelity was studied using a RegenHu 3D bioprinter and comparing SwellMA and SPA to GelMA. Printing measurements were done through three printing tests: (1) one-dimension filament spread, (2) two-dimensions pore size, and (3) three-dimension height and cross-sectional area. 4D bioprinting capabilities were tested through the swelling and shrinking of 3D printed SwellMA constructs.

Results, Conclusions, and Discussions:: SwellMA addresses the need for programmable, high-swelling hydrogels, demonstrating a 500% increase in area, and a 100-fold weight increase (Fig 1B). Additionally, SwellMA demonstrated on-demand shrinking when incubated with CaCl2 solutions, with the biocompatible concentration of 10 mM CaCl2 being the minimum concentration required for maximal shrinking. However, SwellMA was not able to swell back to its initial area after shrinking, which aligns with previously reported limitations of SPA reswelling (Fig 1C). Despite this limitation, the dynamic behavior of SwellMA highlights its potential for 4D bioprinting applications and for investigating the cellular effects of dynamic changes in substrate shape and configuration. SwellMA exhibited a storage modulus of 1.8 kPa, higher than of GelMA and SPA, due to increased covalent bonding within its polymer network. Unlike GelMA, which was fully degraded by day 14, SwellMA resisted enzymatic breakdown by type II collagenase. This resistance to degradation may prove advantageous for applications such as exudate management in chronic wounds with high proteolytic activity. Cell adhesion and proliferation on Swellma were comparable to GelMA, with similar cell counts at day 1 and increased metabolic activity over 3 days, whereas SPA supported minimal cell adhesion (Fig. 1D-E). Interestingly, MSCs in SwellMA exhibited a distinct rounded morphology, unlike the spread morphology in GelMA. This suggests that SwellMA’s resistance to degradation may impair ligand clustering, favoring cell-cell interactions while limiting adhesion and spreading. Notably, MSCs in SwellMA remained metabolically active at rates comparable to GelMA, which is beneficial for tissue engineering applications where a rounded morphology is desirable such as chondrogenesis and adipogenesis. The favorable swelling, rheological, and cytocompatibility properties of SwellMA were leveraged for extrusion-based bioprinting. While SwellMA exhibited lower printing fidelity in filament spread and porosity tests compared to GelMA, its 3D printing performance was superior to SPA, which was non-printable. Additionally, the 3D printed SwellMA constructs were able to maintain their shape during swelling and exhibited controlled shrinking in response to variable osmotic conditions, highlighting its potential for 4D bioprinting and complex shape transformations (Fig 1F-G).

Acknowledgements and/or References (Optional):: T.G.F wants to acknowledge start up funds provided by Lehigh University