(B-66) Additively manufactured polyacrylamide hydrogels exhibit tunable viscoelastic properties that mimic living tissues

Introduction:: Polymerized polyacrylamide (PAA) are linearly elastic hydrogels¹ that are widely used in mechanosensing studies for their biocompatibility, ease of functionalization, and tunable mechanical properties. However, the living tissues are not elastic, but rather viscoelastic¹. As these purely elastic PAA substrates are not suitable for mimicking physiologically relevant viscoelastic microenvironments, here, we developed a stereolithography (SLA)-based additive manufacturing technique to create viscoelastic PAA substrates with tunable mechanical properties and study cellular responses².

Materials and Methods:: We fabricated three PAA substrates of different complex moduli by SLA. Fluorescent markers were added to the acrylamide and bis-acrylamide mixture during the additive manufacturing of the substrates. We evaluated various photoinitiators that are photoreactive around 405 nm due to the system's UV light source requirement and identified TPO-Li to be the most suitable photoinitiator for our purpose. Additional parameters such as the time of exposure (45-60 s) for each layer and layer height (30- 150 μm) were also varied to individually control the G’ and G’’ properties of the substrates. Substrates were functionalized with cyclo-RGDfK peptides to promote cell adhesion. Single live cells were seeded on these dissipative PAA substrates. Early cell spreading behavior together with cell traction and cytoskeletal prestress were evaluated.

Results, Conclusions, and Discussions:: Results: The 3D-printed viscoelastic substrates show a homogeneous and uniform structural composition throughout which the conventional manufacturing techniques cannot produce. The hydrogel porosity can be precisely controlled during 3D printing. Depending on the pore size of the hydrogel, the physical property such as swelling of the hydrogel was also controlled. Rheological investigations show a viscoelastic behavior with a 5−10% loss moduli compared to their elastic moduli, which mimics living tissues. AFM studies showed similar topographical features observed in conventionally-made PAA hydrogels, and thus should not negatively impact living cell studies. When single live cells were seeded on these viscoelastic substrates of varying substrate moduli, we observed an increase in cellular traction and prestress with the increase in substrate stiffness, but not cell spreading. This suggests that cell spreading can be decoupled from traction and intracellular prestress in physiologically relevant environments, unlike the cellular response on elastic conventionally made hydrogels.

Discussions: Conventionally made crosslinked PAA substrates are linearly elastic while the additively manufactured PAA substrates behave as dissipative solids. With additive manufacturing, we can control the dissipative G’’ features by controlling the printing parameters. The source of viscoelasticity can be attributed to entrapped semi-cured and uncured viscous domains within an elastic crosslinked network. This was very evident when the layer height of the gels was precisely controlled. Early cell spreading on the additively manufactured viscoelastic PAA substrates of different stiffness remained almost similar, but cells were able to increase traction and cytoskeletal prestress with increasing substrate moduli, indicating a decoupling of cell spreading from cell traction and cytoskeletal prestress.

Conclusions: We demonstrate a novel method to create viscoelastic PAA hydrogels that mimic living tissues to show that single living cells exhibit sensitivity to the dissipative component of hydrogels. Additive manufacturing provided a lot of control over the shape, size, and mechanical properties of hydrogels. Decoupling cell spreading from traction and prestress is a novel finding which has not been reported in studies conducted on purely elastic PAA substrates. Future studies will focus on further tuning the material properties to study cell fate decisions in disease and development.

Acknowledgements (Optional): : 1. F.C. acknowledges the support of NIH grant 1R15GM140448.
2. S.N. would like to acknowledge NSF grant # 2138459.

References (Optional): : 1. Charrier et al., Nat Commun, 2018.
2. Protick et al., ACS Omega, 2022.