Poster N20 - Dynamically Tunable Acellular Living Hydrogels

Introduction: Engineering dynamically responsive hydrogels that mimic the mechanical properties of tissues may enable living soft materials. The majority of native mammalian tissues comprise non-living extracellular matrices (ECM) and living cell ensembles. ECM dynamically respond to external physical stimuli and stiffen under shear. Non-linear shear-stiffening mechanics are crucial for the tissue structural support and cell functions, such as adhesion, migration, and differentiation. Tailoring biopolymer concentration has been the main approach to tune the non-linear mechanics of ECM-mimetic soft materials, which may compromise other hydrogel properties, including mechanical strength, swelling behavior, degradation rate, and biocompatibility. Additionally, synthetic hydrogels that mimic the ECM mechanical properties may be undermined by the concerns associated with biocompatibility, immune response, and toxic degradation byproducts. As a mechanical-biological trait, self-healing is also highly desirable in fabricating living hydrogels to enable autonomous morphological and mechanical restorations following reoccurring defects. Developing multifunctional living hydrogels with dynamic ECM mechanics and self-healing properties remains an ongoing challenge. In this study, we hypothesized that chemically tailored hairy cellulose nanocrystals (HCNC), bearing a combination of carboxylate and aldehyde moieties on the hairs, can link biopolymer chains in a nanocomposite hydrogel (NCH) matrix via reversible ionic and covalent bonds, resulting in controlled strain-stiffening and self-healing capabilities (Figure 1A). These bifunctional, HCNC linkers are called nLinker, which may render conventional hydrogels living.

Materials and

Methods: To develop acellular nanocomposite living hydrogels (LivGels), dihydrazide-modified alginate (DH-ALG) was synthesized as the core matrix material to offer two types of active sites, i.e., carboxylate groups and hydrazide, for dynamic ionic and covalent bonds, respectively. Anisotropic nLinker, bearing aldehyde and carboxylate binding motifs, were also developed to induce dynamic properties to conventional ALG hydrogels and enable precise control over these properties. The network formation in polymer-polymer or nlinker-nlinker inclusions was tailored via either varying nLinker (integrating only reversible covalent bonds) or calcium ions (Ca2+)-enriched nLinker (integrating both reversible ionic and covalent bonds) concentrations. The mechanical properties of NCH were thoroughly characterized to evaluate the strain-stiffening behavior and as well as self-healing capability. To quantify non-linear stiffening, differential shear modulus, K′ = ∂σ/∂γ, where σ and γ are shear stress and strain, respectively, was calculated for the LivGels. The stiffening index (m) and critical stress (σc) as indicators of the stiffening intensity and the onset of nonlinear regime, respectively, were obtained and compared with other existing dynamically responsive hydrogels. Additionally, the extracted exponents were used to show how the LivGels constituents (i.e., nLinker or Ca2+-loaded nLinker) and the corresponding networks affect the transition between the stiffening mechanisms (i.e., network fluctuation and single filament stretching).

Results, Conclusions, and Discussions: In the LivGels, nLinker form dynamic hydrazone bonds as a result of the Schiff base reaction between nLinker aldehyde groups and ALG-DH hydrazide (Figure 1A). By increasing the nLinker concentration at a constant ALG-DH concentration, the storage modulus increased in the linear viscoelastic (LVE) regime (Figure 1B) and LivGels became stiffer as a result of the increased number of hydrazone bonds and nLinker-mediated network reinforcement increased LivGels stiffness. The mechanical properties of NCH depend on the connectivity between polymer networks and particles. By increasing the Ca2+ concentration from 0 to 36 mM (Figure 1C), the stiffness of LivGels increased because of the additional ionic bonds in the existing network of dynamic covalent hydrazone bonds. With increasing nLinker (Figure 1D) and/or Ca2+-loaded nLinker (Figure 1E) concentrations, the m of LivGels decreased which may be a result of a higher crosslinking density mediated by hydrazone and/or ionic bonds. The denser livGels networks and increased confinement of the polymers by nLinker inclusions effectively limited the stiffening response of the LivGels. The σc of livGels depended on the crosslinker density which was proportional to nLinker or Ca2+ concentrations. The closer the m to 0.5, such as in the case of LivGels including 1.75 wt.% nLinker loaded with 18 mM Ca2+, the filament-mediated stretching would be the dominant mechanism. However, when m approaches 1.5, the bending resulting from network fluctuation preeminently regulates the stiffening responses. By applying a 500% strain, all LivGels indicated fluid-like, viscous behavior detected by storage modulus (G′) < loss modulus (G′′), followed by recovery within seconds (Figure 1F). Together, the non-linear stiffening index (~ 0.09 < m < ~ 0.9), critical stress (~ 7 < σc < ~ 290 Pa), and G′ (from ~ 30 to ~ 1700 Pa) of LivGels were engineered within the biological window, without changing the matrix biopolymer concentration. Overall, this work presents a novel bio-based nanotechnology for engineering acellular hydrogels that mimic ECM, which may enable tissue-mimetic functional biomaterials.

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