Design and Fabrication of Viscoelastic Hydrogels as Extracellular Matrix Mimicry for Cell Engineering

Abstract

The extracellular matrix (ECM) performs both as a static scaffold and as a dynamic, viscoelastic milieu that actively participates in cell signaling and mechanical feedback loops. Recently, biomaterials with tunable viscoelastic properties have been utilized to mimic the native ECM in the fields of tissue engineering and regenerative medicines. These materials can be designed to support cell attachment, proliferation, and differentiation, facilitating the repair or replacement of damaged tissues. Moreover, viscoelasticity modulation of ECM mimicry helps to develop therapeutic strategies for diseases involving altered mechanical properties of tissues such as fibrosis or cancer. The study of biomaterial viscoelasticity thus intersects with a broad spectrum of biological and medical disciplines, offering insights into fundamental cell biology and practical solutions for improving human health. This review delves into the design and fabrication strategies of viscoelastic hydrogels, focusing particularly on two major viscoelastic parameters, mechanical strength and stress relaxation, and how the hydrogel mechanics influence the interactions between living cells and surrounding microenvironments. Meanwhile, this review discusses current bottlenecks in hydrogel-cell mechanics studies, highlighting the challenges in viscoelastic parameter decoupling, long-term stable maintenance of viscoelastic microenvironment, and the general applicability of testing standards and conversion protocols.

1. Introduction

Viscoelasticity is a mechanical property that exhibits both elastic and viscous behaviors.¹ Elastic materials return to their original shape after deformation, while viscous materials dissipate energy during deformation.^2,3 Biological tissues, such as cartilage, muscle, and skin, exhibit viscoelasticity because they possess both spring-like elasticity and fluid-like flow properties.⁴ This unique property enables biological tissues to withstand and dissipate various types of forces while maintaining their structural integrity.⁵ Specifically, the viscoelasticity of biological tissues can be defined in terms of the Young’s moduli or storage moduli (elastic component) and loss moduli, stress relaxation time and creep behaviors (viscous component).^3,6 The viscoelasticity of biological tissues primarily originates from the physical properties and structural composition of their extracellular matrix (ECM).⁷ The ECM is a complex network composed of various biomolecules, including collagen, elastin, proteoglycans, glycoproteins, and noncollagenous proteins.^5,7 Together, these molecules form a dynamic three-dimensional scaffold that provides mechanical strength and plasticity. Among these components, numerous dynamic bonds and physical interactions contribute significantly to the viscoelastic properties.^5,8,9 The degree of cross-linking within the ECM also influences its viscoelasticity, with higher cross-linking enhancing the elasticity and increased cross-linking dynamics promoting viscoelastic behavior.¹⁰ The viscoelasticity of the ECM plays a crucial role in regulating cellular behaviors, including cell sensing, adhesion, migration, proliferation, and differentiation.¹¹

Hydrogels, synthesized from small molecules or polymers known as gelators,^3,12 belong to a sophisticated class of soft, hydrated materials. Their structural integrity is achieved through a network of cross-links, which can manifest via noncovalent interactions such as hydrogen bonds, donor–acceptor pairings, metal ion coordination, or host–guest chemistry, alongside reversible covalent bonds including Schiff bases, borate esters, and disulfide linkages.¹²⁻¹⁴ This dual bonding mechanism endows hydrogels with a nuanced viscoelastic profile resembling the dynamic mechanical properties of biological tissues. The viscoelasticity of hydrogels, defined by their proficiency in absorbing, storing, and dissipating mechanical energy, bestows them unparalleled versatility in biomedical applications. For example, viscoelastic hydrogels well modulate the biophysical and biochemical microenvironments of wound bed during tissue repairing,^2,10,15 which impose a significant influence over cellular behaviors and accelerate the healing process with precision. With the booming development of cell mechanics on viscoelastic hydrogels, innovative applications in cell culture, biofabrication, and the engineering of artificial organs/organoids have been unveiled. These materials stand out as a beacon of hope for developing personalized, functional medical solutions encapsulating the delicate equilibrium between material science and biological necessity.

In this review article, we start with the fundamental introduction of hydrogel viscoelastic parameters (e.g., storage modulus, loss modulus, Young’s modulus, stress relaxation, creep, etc.) and corresponding characterization methods. Subsequently, we discuss the impact of different construction and microenvironment factors on the viscoelasticity of hydrogels. The design and fabrication of viscoelastic hydrogels with various dynamic cross-links (covalent/noncovalent bonds) are then presented to give an overview of reported viscoelastic hydrogel biomaterials for cell mechanics. We then proceed to illustrate how the modulation of viscoelastic parameters, singly or synergistically, can profoundly influence cellular activities and determine cell fate. This exploration underscores the pivotal role that viscoelasticity plays in steering biological responses at the cellular level, thereby highlighting its significance in the development of advanced biomaterials. Given the burgeoning potential of viscoelastic hydrogels in the realms of tissue engineering and biomedical applications, we hope this review offers valuable insights and support for researchers in the field.

2. Characterization and Measurement of Hydrogel Viscoelasticity

The viscoelasticity of hydrogels is determined through a suite of parameters, mainly including Young’s modulus, storage modulus, loss modulus, stress relaxation, and creep behaviors. These parameters are crucial for understanding how the material responds to applied forces and recovers after deformation. A variety of sophisticated instruments are utilized for the characterization of these properties, e.g., nanoindenter, rheometer, atomic force microscope (AFM) and so on.^6,16 Storage modulus and loss modulus refer to the in- and out-of-phase characteristics of a material under stress. The magnitude of their values is affected by various factors, therefore, one should be careful when performing statistical analysis. Taking the rheometer test as an example, the storage modulus and loss modulus depend not only on the cross-linking density and cross-linking types of the material itself but also on the test parameters, such as the thickness of the test sample, the gap between the parallel plates, the test temperature, and the shear frequency. Also, the linear viscoelastic range (LVR) should be identified before testing, typically using the oscillation amplitude mode. In this mode, a strain sweep (usually 0.01–10% for hydrogels) is conducted at a specific angular frequency (usually 1 Hz or 10 rad/s). The LVR is the region where the storage modulus (G′) and loss modulus (G′’) remain constant as the strain is varied. Only within the LVR do G′ and G′’ have clear physical interpretations. Theoretically, the LVR can also be determined using a stress sweep, but this approach is not recommended due to the difficulty in interpreting the results. Two commonly used modes for identifying the structural information and dynamic behavior of hydrogels are the oscillation frequency mode and oscillation time mode. In rheological studies (Figure 1a, b), network systems are classified as hydrogels when 1) G′ > G′′ in both frequency and time mode, and 2) G' and G'' varies with frequency during frequency sweep. The magnitude of G′ represents the stiffness of the hydrogel, with higher values indicating stiffer hydrogels.^6,16

Figure 1.

(a) Frequency sweeps revealed storage (G′) and loss (G″) moduli for viscoelastic hydrogels. (b) Time sweeps revealed storage (G′) and loss (G″) moduli for viscoelastic hydrogels. (c) Applying a constant strain during a stress relaxation test. (d) Applying a constant stress during a creep test.

Stress relaxation and creep tests are two commonly used techniques to characterize the viscoelastic properties of materials and tissue. Stress relaxation measures the decrease in stress over time when a constant strain is applied to the material (Figure 1c). This test is used to characterize the material’s ability to dissipate energy and relax stress, and is more useful for characterizing the short-term viscoelastic properties of materials.^17,18 Creep measures the increase in strain over time when constant stress is applied to a material (Figure 1d). This test is used to characterize the material’s ability to deform under sustained load, and is more useful for characterizing the long-term viscoelastic properties of materials. Quantitative characterization of stress relaxation or creep response is termed τ_1/2 or τ_3/2, which is defined as the time taken for relaxing half of the initial stress value or the time taken for reaching the strain that is 150% of the initial value.^19,20 Considering that stress relaxation (creep), storage modulus, and loss modulus are all affected by material composition, cross-linking methods, etc., decoupling the two parameters to study the effect of a single parameter on cell behavior is also an important and meaningful research direction.

3. Design and Fabrication of Viscoelastic Hydrogels

The viscoelasticity of biological tissues originates from the breakage and formation of weak interactions, the entanglement of polymer chains, and the unfolding of proteins. Similar to biological tissues, the viscoelasticity of hydrogels can also stem from weak physical interactions (such as hydrogen bonds, metal coordination bonds, hydrophobic association, etc.), dynamic covalent bonds (imine bonds, borate ester bonds, thioester bonds, disulfide bonds, etc.), and the entanglement of polymer chains. To construct viscoelastic hydrogels that meet the requirements of biological applications, the choice of main chain and cross-linking method is particularly important.

3.1. Noncovalent Cross-Linking

3.1.1. Hydrogen Bonds

Hydrogen bonds play a significant role in hydrogel construction. Although individual hydrogen bonds are weak, their cooperative action strengthens the intermolecular interactions. By variation of the hydrogen bond parameters, hydrogels with tunable mechanical properties can be created. Moreover, the dynamic nature of hydrogen bonds enables the creation of responsive viscoelastic gels whose rheological properties can be controlled by manipulating factors such as temperature, pH, and ionic strength. This opens up applications in drug delivery, tissue engineering, and biosensing.

Recently, Wu and co-workers²¹ investigated the influence of hydrogen bonds on the dynamics and viscoelastic behavior of gels using poly(acrylamide-co-methacrylic acid) copolymers (P(AAm-co-MAAc)) as a model system (Figure 2a). It is found that the breaking of hydrogen bonds within and between polymer chains dissipates energy, leading to improved mechanical properties (Figure 2b). By incorporating a moderate amount of acrylamide (AAm) units into the Poly methacrylic acid (PMAA) chains, they were able to promote hydrogen bond formation while reducing the charge density and electrostatic repulsion between chains, resulting in enhanced gel stability (Figure 2c). This study provides valuable insights for the design of high-strength gels with excellent performance. Zhu and co-workers²² reported a versatile approach for synthesizing hydrogen-bonding polymeric adhesives with barbiturate (Ba) and Hamilton wedges (HW) as side chains is presented. Rubber copolymers with thiol-reactive groups are modified with Ba and HW via an efficient one-pot procedure, forming hydrogen-bonded supramolecular polymer networks (Figure 2d). The presence of Ba/HW enhances the network integrity, and either Ba or HW alone enables strong adhesion to diverse substrates, surpassing commercial adhesives (Figure 2e, f). H-bonds play a crucial role in improving the mechanical properties and adhesion behavior of the supramolecular polymer, with dynamic association/dissociation facilitating energy dissipation within the material. This work highlights the potential of hydrogen bonds in guiding the self-assembly of polymeric networks for the development of versatile, reversible, and self-healable adhesives. Zhao²³ proposed a “time-salt type” superposition principle by combining the Hofmeister effect with the “time–temperature superposition” from polymer physics (Figure 2g). This principle explains the significant changes in the viscoelasticity of poly(methacrylamide)(PMAm) hydrogels upon the addition of different types of salts. Salting-out ions strengthen hydrogen bonds in the PMAm main chain, leading to a denser cross-linked structure that restricts water movement. Conversely, salting-in ions promote hydrogen bonding between water and amide groups, disrupting chain hydrogen bonding and accelerating relaxation. This reduces the restriction of the gel network on the water movement. The study enables the control of hydrogel viscoelasticity via the Hofmeister effect. The self-assembling peptide hydrogels, also primarily formed through hydrogen bonding, possess several notable advantages.²⁴ First, these peptides often originate from biomacromolecules, making them more compatible with biological systems. Second, by adjusting parameters such as the peptide sequence and solution concentration, one can effectively control the properties of the hydrogels, including their mechanical strength, pore structure, and swelling behavior (Figure 2h). Additionally, due to the simple chemical structure of the peptides, their preparation process is relatively straightforward and cost-effective. In terms of viscoelastic regulation, peptide-based hydrogels can adjust their viscoelastic properties by altering their peptide sequences or concentration. For example, introducing hydrophobic amino acid residues can make the gel structure denser, thereby enhancing its mechanical strength and stability; whereas increasing the number of hydrophilic amino acid residues makes the gel softer and more pliable. Moreover, the viscoelasticity of the gels can also be modulated by adding other substances such as polyelectrolytes or polysaccharides. In recent years, research teams led by Yan have utilized short peptide self-assembly technology to develop a series of hydrogels with excellent viscoelastic characteristics.²⁵⁻²⁷ These have been successfully applied in areas such as immune regulation, tumor treatment, and antibacterial applications, showcasing broad prospects for the use of hydrogel materials in the medical field.

Figure 2.

(a) Schematic representation for the network structure of the tough gels with hydrogen bond associations in the nonstretching state (left) and during stretching (right). (b) Tensile stress–strain curves of AM-0.25–6 hydrogels with a water content of 39 wt % at different deformation rates. (c) Corresponding mechanical properties of AM-0.25–6 hydrogels with a water content of 39 wt % at different deformation rates. (d) Schematic representation of the dynamic interfacial gluing between P(nBuA-co-Ba-co-HW) and substrate through diverse H-bonding. (e) Stress–strain curves for P(nBuA-co-Ba-co-HW) as adhesives at a debonding speeds of 10, 100, 1000 μm·s^–1 at 25 °C. (f) Stress–strain curves for P(nBuA-co-Ba-co-HW) as adhesives at 25, 60, 90 °C with debonding speeds of 100 μm·s^–1. Reproduced with permission from ref (22). Copyright 2022 WILEY-VCH. (g) Illustration of the salt effect on the H-bonding cross-linked in PMAm hydrogel. (h) Swelling volume ratio (Qv) of PMAm hydrogels soaked in various salts aqueous solutions (1.0 M) relative to the H₂O-equilibrated PMAm hydrogel.

3.1.2. Hydrophobic Associations

Hydrophobic association refers to the interaction between hydrophobic molecules, which leads to their aggregation and the formation of organized structures in water. This phenomenon has significant applications in the construction of viscoelastic hydrogels. For example, Baker and Moroni²⁸ successfully achieved molecular tuning of self-assembled supramolecular benzene-1,3,5-tricarboxamide (BTA) hydrogels by making simple adjustments to hydrophobic substituents. As a result of this tuning, the fiber hydrogels exhibited stress relaxation changes that exceeded 5 orders of magnitude, while maintaining a relatively constant storage modulus. Additionally, all hydrogels in this series were injectable and showed excellent shape fidelity and stability after 3D printing. Notably, BTA hydrogels exhibited shear thinning and self-healing properties, while retaining their ECM-mimicking viscoelastic and fibrous characteristics. Grunlan’s group²⁹ has developed a triple network (TN) hydrogel that utilizes electrostatic and hydrophobic interactions to enhance its modulus (Figure 3a). The TN hydrogel consists of three interconnected networks: an anionic first network, a neutral second network capable of hydrophobic association, and a cationic third network. By adjustment of the concentration of the cationic third network, the TN hydrogel can achieve compressive moduli similar to those of articular cartilage (∼1.0 MPa), temporomandibular joint disc (TMJ) (∼2.0 MPa), and intervertebral discs (IVD) (∼3.0 MPa) (Figure 3b). Additionally, the TN hydrogel exhibits tensile moduli comparable to those of tracheal cartilage (∼2.0 MPa) and IVD annulus fibrosus (∼2.5 MPa) (Figure 3c).

Figure 3.

(a) TN networks were formed with an anionic first network, a neutral second network, a cationic third network, and the resulting “TN-APATAC” hydrogels. (b) Compressive modulus of TN-APTAC hydrogels. (c) Tensile modulus of TN-APTAC hydrogels. Reproduced with permission from ref (29). Copyright 2022 WILEY-VCH. (d) Schematic representation of hydrogel formation utilizing functionalized 4 arm PEG, PEG-FGG (red), and PEG-SB (yellow). Molecular structure of the guest FGG and stilbazolium iodide. (e) Frequency sweeps of 3.5 wt % IPN hydrogels with 0%, 25%, 50%, 75%, and 100% PEG-SB after 10 min irradiation with a 370 nm light source. (f) Stress relaxation of 3.5% hydrogels composed of single network (SN) or interpenetrating network (IPN). Reproduced with permission from ref (30). Copyright 2024 WILEY-VCH. (g) Schematic illustration of the preparation of supramolecular hyaluronic acid hydrogels stabilized by different pairs of host–guest complexation and the monitoring of 3D spreading of encapsulated hMSCs. (h) Swelling ratio of the supramolecular hydrogels measured after incubation in culture medium for 3 days. (i) Average value of G′ and G″ from rheological analysis at the frequency of 0.1 Hz and 1% strain of the supramolecular hydrogels. (j) Different staining of hMSC-laden hydrogels of different groups after 14 days of osteogenic culture. Reproduced with permission from ref (32). Copyright 2021 Springer Nature.

Wang³⁰ demonstrated an innovative design called an interpenetrating network (IPN) using the host–guest interaction between cucurbit[8]uril (CB[8]) and star-shaped poly(ethylene glycol)s functionalized with Phe-Gly-Gly tripeptide or photoactive stilbazolium as guest molecules (Figure 3d). First, by simply mixing the two polymers in an aqueous medium, a physically cross-linked network with fast relaxation is formed in the initial stage. Then, through external light stimulation, the network can rapidly and selectively convert to a robust covalent network. This unique capability allows for precise control over the physical and chemical cross-linking density, enabling accurate modulation of the hydrogel’s stiffness, stress relaxation, and self-healing properties (Figure 3e and 3f). Also, Bian’s team³¹⁻³³ has proposed a Host–Guest-Macromer (HGM) strategy in recent years, dedicated to the development of novel dynamic hydrogels and their applications in stem cell culture and tissue engineering (Figure 3g). It was found that biomacromolecules cross-linked by multiple host–guest interactions possessed a suitable viscoelasticity for long-term stem cell culture and research. Moreover, affected by the dynamic behavior of host–guest interactions (i.e., host–guest inclusion complexation rate constant), this kind of cell-compatible three-dimensional hydrogel exhibited rapid mechanical dissipation (Figure 3h and 3i), injectability and reshaping, which facilitated the cell differentiation (Figure 3j).

3.1.3. Ionic Bonds and Metal–Ligand Bonds

Ionic bonds and metal–ligand bonds are two distinct types of chemical bonds that differ significantly in their formation mechanisms, structural features, and physicochemical properties. Ionic bonds are dominated by electrostatic interactions.³⁴ In contrast, metal–ligand bonds are polar covalent bonds that involve a central metal atom or ion (known as the coordination center) and at least one ligand that contains lone pairs of electrons. Both types of bonds find applications in the construction of viscoelastic gels.

Li³⁵ developed a two-step equilibration method to enhance the mechanical properties of polyampholyte (PA) gels (Figure 4a). The method involves dialyzing the PA gel against a multivalent metal ion solution and then against deionized water (Figure 4b, c). This approach significantly improves the Young’s modulus (Figure 3d), tensile fracture strength (Figure 4e) and tearing energy of the PA (Figure 4f, g) gel compared to the pristine gel. The method can also be generalized to different PA gels and multivalent metal ions. The resulting hydrogels exhibit good signal transduction due to their stable ionic conductivity, showing potential as strain sensors (Figure 4h, i). The contribution of viscoelasticity and elasticity to the mechanical properties of the hydrogels was also discussed via a viscoelastic model to further understand the strengthening and toughening mechanisms.³⁶ In 2013, Gong’s group reported polyampholyte hydrogels (PA hydrogels) with fracture energy of 1000–4000 Jm^2–, adjustable modulus of 0.01–8 MPa, and fracture strain of 150–1500%. These hydrogels exhibit nearly 100% self-healing ability.³⁷ Their high toughness originates from synergistic energy dissipation of a multiscale structure.³⁸ The sources of energy dissipation in the multiscale structural system, from smallest to largest scales, are as follows: at the molecular level (energy dissipation due to ionic bonds formed by positive and negative ions), at the nanoscale (energy dissipation due to molecular chain segments), at the microscopic level (energy dissipation due to the dual-network structure), and at the macroscopic level (energy dissipation due to the hydrogel topology). During deformation, the microscopic double network undergoes sequential affine and nonaffine deformations. During affine deformation, the double network remains intact but collapsed molecular chains stretch and dissipate energy via ionic bond breakage. After the affine deformation, the high-modulus network starts to break. The low-modulus network then plays two roles: stress transfer and stress concentration reduction, preventing rapid crack propagation and sample failure. Further stretching destroys the low-modulus network, leading to macroscopic fracture. This multiscale damage coupling results in multiscale energy dissipation and high toughness. Two parameters are crucial: the relative strength of the high- and low-modulus networks and the absolute strength of the ionic bonds. The former affects stress transfer and concentration reduction, while the latter affects energy dissipation and viscoelasticity.³⁷

Figure 4.

(a) Design of the strong tough polyampholyte (PA) hydrogels via the synergy of ionic and metal–ligand bonds. (b) Chemical structures of the monomers in the strong tough PA gels. (c) Possible dynamic bonds formed in the strong tough PA gels. (d) Young’s modulus of the samples. (e) Fracture strength of the samples. (f, g) Tearing force versus displacement curves of the samples and corresponding tearing energy versus FeCl₃. (h) Complete circuit composed of a LED bulb and the gel sample, demonstrating the brightness of LED response to applied strains (i–iv) on the sample under loading–unloading cycles. (i) ΔR/R₀ versus applied time of the hydrogel sensor on wrist. Reproduced with permission from ref (35). Copyright 2021 WILEY-VCH.

3.2. Dynamic Covalent Cross-Linking

A dynamic covalent bond is a type of covalent bond that can reversibly break and form under certain conditions. In principle, all chemical reactions exhibit some degree of reversibility. However, the extent of reversibility varies significantly due to differences in chemical bond energies (e.g., covalent bonds typically have energies of 150–800 kJ/mol, coordination bonds, 80–350 kJ/mol, and hydrogen bonds, 0–20 kJ/mol). The reversibility of a covalent bond can be quantified using the thermodynamic equilibrium constant (K_θ) of its bond formation reaction. When K_θ falls between 1 × 10⁷ and 1 × 10^–7, the reaction is considered reversible. Reactions with K_θ values greater than 10⁷ or less than 10^–7 are considered irreversible. Dynamic covalent bonds play a crucial role in the viscoelastic properties of many biological materials such as proteins and polysaccharides. These bonds allow for the reversible rearrangement and reorganization of molecular structures, which contributes to the materials’ ability to adapt to mechanical stress and maintain their integrity. For instance, in the extracellular matrix (ECM) of cells, dynamic covalent bonds between collagen and elastin proteins (primarily involving aldimine and ketoneimine bonds between lysine and hydroxylysine residues) provide viscoelasticity to the tissue. These bonds allow the ECM to withstand mechanical forces and recoil to its original shape after deformation, as far as possible.

3.2.1. Imine Bonds

Three-dimensional bioprinting has emerged as a promising tool to spatially pattern cells to fabricate model tissues of the human body. Recently, Heilshorn and colleagues³⁹ introduced an engineered bioink material whose viscoelastic mechanical behavior could be tuned to resemble that of living tissue (Figure 5a). The gel contains a dynamic covalent bonding network based on hydrazone linkages, allowing the material to undergo spontaneous breakage and reformation under physiological conditions. To overcome the erosion and viscous flow issues associated with dynamic cross-linking during the printing process, the authors also developed a strategy to dynamically modulate the cross-linking kinetics and network formation using small molecule competitors and catalysts, a glycine-based hydrazide analogue (that disrupts hydrazone bond formation) and a benzimidazole sulfonated derivative (that accelerate hydrazone bond formation), respectively. These diffusible small molecules allowed for independent tuning of the network dynamics before and after printing, leading to a bioink that has a printable viscoelasticity initially and that can be matured to a desired viscoelasticity for long-term cell culture. Xia⁴⁰⁻⁴² designed a series of dynamic covalent hydrogels cross-linked via hydrazone bonds while keeping the cross-linking density constant by varying the concentration of the organic catalyst to tune the exchange rate of hydrazone bonds. This strategy allows for analysis of the viscoelastic response of dynamic hydrogels as a function of their network parameters (Figure 5b). They found that the terminal relaxation time of dynamic hydrogels is primarily determined by two factors: the cross-linking exchange rate and the effective number of cross-links per polymer chain. A universal correlation exists between the terminal relaxation time and the exchange rate, which is in principle extendable to any viscoelastic hydrogel network. This quantitative relationship aids the development of dynamic hydrogels with tunable desired viscoelastic responses based on molecular design. Using aminomethyl benzimidazole as the organic catalyst accelerates the dynamic exchange of hydrazone bonds.⁴³ The addition of the catalyst reduces the activation energy of the exchange reaction without affecting its equilibrium constant. Therefore, it is reasonable to assume that changing the amount of catalyst can continuously adjust the exchange kinetics of the cross-linking without changing the cross-linking density at equilibrium. This decouples the cross-linking kinetics and cross-linking density of the viscoelastic hydrogels. Gerecht⁴³ engineered a viscoelastic hydrogel system via dynamic covalent cross-linking (Figure 5c). The gel is composed of gelatin and dextran, with gelatin as the major component. Dynamic covalent bonds, imine and acylhydrazone, form the dynamic network hydrogels (denoted as D-hydrogels), with static covalent bond methacrylates (MAs) forming the control static hydrogels (denoted as N-hydrogels). The researchers not only controlled the stiffness of the hydrogels by controlling the cross-linking conditions but also evaluated the mechanical strength, stress relaxation, and other parameters of the hydrogels before and after cell culture with culture medium, respectively. This study identified the role of the dynamic network matrix and its potential mechanism in regulating the formation of vascular tissue morphology; i.e., the dynamic hydrogel network promotes the formation of large focal adhesions by enhancing cell contractility, recruiting adhesion proteins, and promoting the formation of large focal adhesions, thus rapidly generating vascular networks.

Figure 5.

(a) Schematic of the engineered HELP bioink, which consists of HA modified with either an ALD or BZA group and an ELP modified with a HYD group. Reproduced with permission from ref (39). Copyright 2023 The American Association for the Advancement of Science. (b) Schematic representation of two polymer network architectures: cross-linking of linear polymers and end-linking of star-shaped polymers functionalized with hydrazine and aldehyde. With the catalyst used to accelerate hydrazone exchange. Reproduced with permission from ref (42). Copyright 2021 WILEY-VCH. (c) Engineered viscoelastic hydrogels with dynamic cross-links permit cell contractility-mediated integrin clustering and FAK activation, independent of hydrogel stiffness, and promote vascular assembly. Reproduced with permission from ref (43). Copyright 2020 Elsevier.

3.2.2. Disulfide Bonds

In dissociative networks, the stiffness, a property dictated by the cross-link density, is related to the cross-link equilibrium constant (K_eq = k_a/k_d), whereas stress relaxation is related to the cross-link dissociation rate (k_d). Thus, changing k_d typically results in concomitant changes in the characteristic time scale of stress relaxation (τ) and the initial modulus (G₀) that describes the stiffness. To achieve decoupling of the two parameters, the group of Kalow,⁴⁴ inspired by the “declick” reactions developed by Anslyn and co-workers,⁴⁵ developed glassy polydimethylsiloxane (PDMS) materials using thiolene and thiolyne conjugate addition–elimination reactions for cross-linking. The research shows that by using different conjugate acceptor cross-linkers, the relaxation rate of the glassy material can be adjusted over 4 orders of magnitude without affecting its stiffness. This principle is also applicable to gels, where viscoelastic decoupling can be achieved. This mechanistic insight into cross-link exchange and parametrization of cross-link reactivity enable the design of materials with targeted viscoelasticity. Recently, Otto’s group⁴⁶ reported a new dynamic covalent hydrogel system based on peptides featuring two orthogonal reversible chemistries, enabling distinct functionalities. Dynamic disulfide bonds allow for the self-templated synthesis of macrocycles that undergo supramolecular polymerization. Bioorthogonal dynamic hydrazone chemistry enables the covalent functionalization of the resulting fibers from both the cross-linker and with biologically relevant ligands. The resulting material features viscoelastic properties similar to those of the extracellular matrix. Moreover, the hydrogel allows for the simple and on-demand attachment of biologically relevant ligands or known short peptide sequences, guiding stem cell differentiation. Due to its peptide nature, the material is inherently cytocompatible (Figure 6a). Dai’s group⁴⁷ fabricated a hydrogel network by employing silver sulfadiazine (AgSD) as a catalyst to introduce both imine bonds and disulfide bonds onto the same cross-linking chain (Figure 6b). Notably, AgSD facilitates the formation of imine bonds within the hydrogel, thereby bolstering its stability over a specific time frame. Additionally, the silver ions present in AgSD establish Ag–S coordination with the disulfide bonds on the same cross-linking chain, augmenting the hydrogel’s dynamic properties. This ingenious approach effectively reconciles the stability-dynamics paradox commonly associated with imine bond hydrogels. Furthermore, AgSD, being an antibacterial agent, imparts significant bactericidal capabilities to the hydrogel, which aids in the healing of bacteria-infected wounds. Controlling dynamic parameters (creep rate, stress relaxation rate, and self-healing rate) is crucial for designing viscoelastic materials for various applications. This requires understanding how molecules contribute to the dynamic properties of the materials.⁴⁸

Figure 6.

(a) Novel peptide-based dynamic covalent hydrogel system with two orthogonal reversible chemistries that perform three different roles. Mechanism of fiber formation starting from peptide functionalized, aromatic dithiol and the schematic representation of the stepwise hydrogelation process, with hydrogel formation upon the addition of different cross-linkers. In panel (a), ①–⑥ represent in sequence: oxidation of thiois to cyclic disulfides; exchange; nucleation; elongation; fragmentation; hydrogelation. Reproduced with permission from ref (46). Copyright 2023 WILEY-VCH. (b) Schematic diagram of the synthesis and properties of OHAC hydrogels. AgSD accelerated the formation of imine bonds and enhanced the network stability, network dynamics, and antibacterial properties of hydrogels. Reproduced with permission from ref (47). Copyright 2024 WILEY-VCH.

3.2.3. Borate Ester Bond

Cells can sense variations in the stiffness and viscous properties of their surrounding microenvironment and use these cues to adapt their gene expression in response to their surroundings. While the time scales over which cells perform mechanosensing are not well understood, hydrogels with tunable viscoelastic properties can be used to probe cellular responses to the mechanical properties of their surroundings in a time-dependent manner. Cooper-White et al.⁴⁹ reported on a viscoelastic hydrogel culture system based on the reversible borate cross-linking of pendant boronic acids and diols (Figure 7a), where the viscoelastic properties could be modulated by exploiting the equilibrium dynamics of the esterification reaction. This cellular effect could also have applications in wound healing.

Figure 7.

(a) Design of adaptable hydrogels via reversible boronate esterification and rheological characterization. Reproduced with permission from ref (49). Copyright 2019 Elsevier. (b) Schematic illustrations of PVA/BA adhesives. Instant softening of the top near-surface region upon water activation allows for fast adhesion to the wound site through dynamic covalent bonds. Reproduced with permission from ref (50). Copyright 2022 National Academy of Sciences. (c) Representative images of skin wound tissues on days 3, 7, and 15 following immunofluorescences labeling of tumor necrosis factor-α (red) and representative images of the skin wound tissues on days 7 and 15 following immunofluorescence labeling with vascular endothelial growth factor (red). Reproduced with permission from ref (51). Copyright 2021 WILEY-VCH.

A rapidly reversible adhesive composed of dynamic borate ester bonds, formed from poly(vinyl alcohol) (PVA) and boric acid (BA), has been developed by Shu and colleagues⁵⁰ to use as a wound dressing adhesive (Figure 7b). The adhesive can achieve a bonding shear strength of 61 N/cm² and a transdermal bonding strength of 511 N/cm² within 2 min. The bonded layer can be debonded readily upon exposure of the adhesive to excess water. The adhesive exhibits strong adhesion to diverse substrate surfaces regardless of whether the surfaces are smooth (e.g., glass) or rough (e.g., rat fur), and the PVA/BA adhesive outperforms the most widely used skin adhesives in clinical use. A series of boronic acid dynamic covalent bonds composed of C = C double bonds were developed by Chen et al.,⁵¹ with which an injectable hydrogel dressing was constructed with rapid gelation and on-demand dissolution properties. The hydrogel was fabricated by mixing 2-formylphenylboronic acid (2-FPBA), cyanoacetate-terminated 4-arm PEG (4armPEG-CA), and poly(vinyl alcohol) (PVA). The boronic acid and cyanoacetate underwent a rapid Knoevenagel condensation (CKC) reaction to form dynamic double bonds, thus forming a hydrogel (2-FBC). In the presence of cysteine (Cys), the hydrogel rapidly dissolved due to the formation of thiazolidine boronic acid boronate (TzB) complexation with the components in the hydrogel. As a result, the dressing replacement would attenuate the secondary trauma to the wound due to the in situ degradation of hydrogels (Figure 7c).

4. Viscoelastic Hydrogel Biomaterials for Cell Biology

Cells are highly sensitive to the mechanical properties of their surroundings. Viscoelasticity is a crucial factor that influences various cellular processes, including cell adhesion, migration, differentiation, and proliferation.⁵² Cells can sense viscoelastic cues in their microenvironment through specialized structures such as focal adhesions and integrins⁵³⁻⁵⁵ (Figure 8a and 8b). These structures are connected to the cytoskeleton, a network of protein filaments that provides structural support and enables cells to respond to mechanical cues. When cells interact with a viscoelastic substrate, they exert forces on the substrate, which in turn generates force feedback to the cells.⁵⁶ The cells can sense the viscoelastic properties of the substrate by monitoring the deformation and the rate at which the substrate relaxes after the force application. The physiological mechanisms underlying how cells sense viscoelasticity are still being actively studied, and one classic research model is the molecular clutch model.^57,58 In this model, the adhesion complexes behave akin to a mechanical clutch in an engine, which can engage or disengage the transmission of force from the cytoskeleton to the ECM. When the clutch is “engaged”, myosin-generated forces are effectively transmitted to the ECM, allowing for strong adhesion, traction force generation, and potentially, signaling events that influence cell behavior. Conversely, when the clutch is “disengaged”, force transmission is reduced, facilitating cell detachment or migration.

Figure 8.

(a) The expanding cell monolayer is modeled as a one-dimensional contractile continuum that exerts forces on its underlying deformable substrate through discrete focal adhesions and viscous friction. (b) (Top) Each focal adhesion is modeled as a clutch. Actomyosin-driven contraction of the monolayer causes substrate deformation and actin retrograde flow according to the binding/unbinding dynamics of focal adhesion proteins under force. (Bottom) To maintain force balance across the monolayer after each myosin-driven contraction step, the substrate is pulled by a larger amount on the soft side (d₁) than on the stiff side (d₂), thereby tilting overall expansion toward the stiff side (d_CM = d₁ – d₂). Reproduced with permission from ref (55). Copyright 2016 American Association for the Advancement of Science.

In hydrogel-cell mechanics studies, cell culture methods are divided into 2D and 3D modes. Due to different modes of interaction between cells and the extracellular matrix (ECM), the two methods directly affect force transduction and normal physiological activities of cells. Traditional 2D cell culture involves culturing cells on flat, solid surfaces such as Petri dishes or glass coverslips. Cells contact the matrix only on the bottom surface, forming a fixed adhesion point. In this setting, mechanical stimulation mainly comes from the supporting surface, causing cells to flatten and limiting cell–cell and cell-ECM interactions.⁵⁹ Force transmission occurs primarily from the bottom of the cell inward, potentially leading to incomplete biomechanical sensing. This can impact normal cellular functions such as signal transduction, gene expression, proliferation, and differentiation. Long-term 2D culture may also result in cells losing their natural state and functional characteristics. In contrast, 3D cell culture better mimics the complex in vivo environment by allowing cells to be suspended in a matrix gel or forming aggregates like spheroids, microcapsules, or tissue-like structures. Cells can contact the matrix on multiple sides, increasing the contact area and contact points. The force transmission pathway becomes more intricate, exposing cells to realistic cell–cell and cell-ECM interactions. This enables the formation of 3D structures that resemble the in vivo environment, such as cell polarity, gap junctions, and tissue-specific structures.⁶⁰ By sensing physical forces from multiple directions, cells in the 3D culture maintain their natural morphology and function. This promotes cell-to-cell signaling, enhances differentiation, and more accurately reflects the physiological and pathological states of the tissue. For instance, tumor cells in 3D culture can form a microenvironment similar to actual tumors, exhibiting characteristics like hypoxia and drug resistance, which is crucial for cancer research and drug screening.^61,62

4.1. Regulation of Cells by Varying Single Viscoelastic Parameter

4.1.1. Mechanical Strength or Stiffness

The mechanical strength of a gel has a significant impact on cell migration behavior, known as durotaxis, which refers to the phenomenon where cells tend to migrate toward regions with higher mechanical strength.⁶³ This behavior involves cell hardness sensing, activation of signaling pathways (such as RhoA, Rac1, and Cdc42), and morphological and cytoskeletal reorganization. However, in reality, the term “durotaxis” is not accurate. In 2017, Odde⁶⁴ discovered that many types of cells demonstrate an optimal stiffness at which their migration is maximized. They developed a cell migration simulator that can predict this optimal stiffness, which can be altered by adjusting the number of active molecular motors and clutches. This prediction has been experimentally validated by comparing the cell traction and retrograde flow of F-actin in two different cell types with varying levels of active motors and clutches. And the positive or negative durotaxis of a cell is determined by its contractile and adhesive machinery.⁶⁵ For example, through precise control of the degree of methacrylation of GelMA, Fernandez et al.⁶⁶ successfully achieved the customization of matrix strength. Based on this design, they investigated the impact of mechanical strength on the invasive behavior of breast cancer cells (Figure 9a). The research findings indicated that in soft matrices the cell migration efficiency was higher. And they proposed the concept of a “Goldilocks window” for cells, not durotaxis. In addition to migration, mechanical strength also plays an inductive role in determining the direction of stem cell differentiation. To evaluate the effect of substrate stiffness on stem cell behavior, Lee et al.⁶⁷ used a mixture solution of poly(vinyl alcohol) (PVA) and hyaluronic acid (HA) and employed a gradual freeze method (the solution gradually freezes along the longitudinal direction to the top side) to prepare hydrogels with a wide range of stiffness gradients and cell adhesiveness. Human bone marrow mesenchymal stem cells (hBMSCs) were used as the model cells (Figure 9b). They measured the adhesion and growth of cells on the PVA/HA hydrogels with stiffness gradients by assessing the DNA content. The study found that hBMSCs in softer gels exhibited better neurogenic differentiation (Nestin expression), while stiffer gels induced osteogenic differentiation (osteopontin expression). On the other hand, in myogenic and chondrogenic differentiation, substrates with soft to moderate hardness and moderate hardness, respectively, exhibited significantly higher expression levels of target proteins compared to other substrates (neuronal cells at 20 kPa, muscle cells at 40 kPa, chondrocytes at 80 kPa, and osteogenic cells at 190 kPa).

Figure 9.

(a) MCF-7 and MDA-MB-231 single cell trajectories cultured in GelMA hydrogel variants; tracks were normalized with respect to the origin. Reproduced with permission from ref (66). Copyright 2020 WILEY-VCH. (b) Quantitative real-time PCR analyses after 2 and 4 weeks of hBMSC culture on the PVA/HA hydrogel with a stiffness gradient (n = 3; *p < 0.05). The softer hydrogel sections allowed better neurogenic differentiation than the stiffer sections, while the stiffer hydrogel sections induced higher osteogenic differentiation. Meanwhile, there was greater differentiation into higher muscle and cartilage cells on the hydrogel sections with moderate stiffness (∼40 kPa, for muscle cell; ∼80 kPa, for chondrocyte). Reproduced with permission from ref (67). Copyright 2016 Elsevier. (c) Immunofluorescent images of fibroblasts cultured on glass and PAAm gels of various stiffness for 2 days and stained for F-actin (phalloidin, red), HAT1, and β-actin (green). Scale bars: 50 μm. Reproduced with permission from ref (70). Copyright 2024 The American Association for the Advancement of Science.

Bone cells play a vital role in maintaining bone homeostasis, and their dysfunction can lead to diseases, such as osteoporosis. Current clinical strategies using drugs may disrupt innate bone metabolism. Therefore, there is an urgent need for an alternative strategy to precisely control the differentiation of bone cells. To address this, Liu et al.⁶⁸ proposed the hypothesis that mechanical stimulation could be a potential strategy. They created a hydrogel to simulate the physiological bone microenvironment, with stiffness ranging from 2.43 to 68.2 kPa, and investigated in depth the influence of matrix stiffness on the behavior of bone cells. The results indicate that matrix stiffness can guide the fate of the bone cells. In particular, increasing matrix stiffness inhibited the mechanical transduction pathway related to integrin β3-sensitive RhoA-ROCK2-YAP, and promoted the differentiation of bone cells. Notably, the medium-stiffness hydrogel (M-gel) partially inhibited osteogenic differentiation within the range of 17.5 kPa-44.6 kPa, and subsequently promoted blood vessel regeneration and bone regeneration in mice with bone defects.

Similarly, Weaver’s research⁶⁹ has shown that mechanical strength stimulation of the ECM can modulate cell signaling pathways, affect gene expression, and consequently alter cell behavior. For example, by altering cell signaling pathways, we can increase the ratio of stem progenitor cells in tissues. Furthermore, mechanical strength stimulation of the ECM can also regulate the activity of the progesterone receptor (PR), leading to an increase in the number of normal mammary epithelial progenitor cells. This change may promote the occurrence and development of breast tumors. Li et al.⁷⁰ reported the biphasic regulation of matrix stiffness on epigenetic state during cellular reprogramming (Figure 9c). The study found that during the reprogramming of fibroblasts into neuronal cells on different matrix stiffnesses, fibroblasts cultured on a 20 kPa intermediate stiffness hydrogel showed an increased intracellular G-actin and Cofilin-mediated nuclear transport pathway. This pathway facilitated the translocation of acetyltransferase (HAT) from the cytoplasm to the nucleus and its activation within the nucleus, leading to enhanced chromatin accessibility and ultimately promoting the efficiency of fibroblast-to-neuronal cell reprogramming. However, the reprogramming efficiency of fibroblasts cultured on soft (1 kPa) and hard (40 kPa) substrates shows only modest improvement for neuronal cell conversion.

Furthermore, the mechanical strength also influences the immunophenotype of MSCs. Xu et al.⁷¹ analyzed the mechanical properties of healthy and periodontitis-rat PDL-AB enthesis and the immunophenotype of MSCs, suggesting that the anti-inflammatory phenotype of MSCs (MSC2) is likely related to the stiffness gradient strength of interface tissues (referred to as SGS characteristics). By combining immunofluorescence techniques and mechanical models, it was revealed that SGS characteristics regulated the MSC2 phenotype through integrin and myosin IIB polarization as well as nuclear mechanical transduction.

4.1.2. Stress Relaxation

Currently, cell migration is mainly characterized by the formation of leading edge filopodia, mature adhesion plaques, and dispersal phenotypes, which often occurs in elastic matrices. However, biological tissues are mostly viscoelastic and exhibit stress relaxation properties. Chaudhuri and colleagues⁷² investigated the impact of different stress relaxation matrices on cell migration by cross-linking alginate with various calcium ion cross-linkers and synthesizing an interpenetrating matrix network (IPNs) with reconstituted basement membrane (rBM). The results from the mean square displacement (MSD) and migration velocity of HT-1080 cells showed that cells migrated further and faster on matrices with rapid and intermediate relaxation, compared to the slow relaxation group. Similarly, the cell migration results of MDA-MB-231 cells and MCF-10A cells indicated that cells exhibited a faster migration speed and longer migration distance on matrices with rapid stress relaxation. This study discovered that matrix stress relaxation is a critical factor for cell migration in viscoelastic matrices.

In addition to migration, stress relaxation also plays a regulatory role in stem cell differentiation. The process of cartilage formation by mesenchymal stem cells (MSC) in 3D culture involves dynamic changes in cell cytoskeleton structure during the precondensation phase of morphogenesis. However, the connection between the dynamic mechanical properties of the matrix and the changes in the cell cytoskeleton during cartilage formation remains unclear. Recently, Xiao’s group⁷³ explored how stress relaxation influences the changes in MSC cell cytoskeleton at various stages of cartilage formation (Figure 10a and 10b). The study revealed that in slowly relaxing hydrogels, although disorganized actin promoted early chondrogenesis, prolonged myosin activation resulted in ROCK-dependent cell apoptosis. On the other hand, fast-relaxing hydrogels promoted cell-matrix interactions, facilitating long-term cartilage formation and reducing myosin overactivation and cell apoptosis, similar to the effects of ROCK inhibitors. This study not only sheds light on how matrix stress relaxation regulates MSC chondrogenesis and survival in a ROCK-dependent manner but also emphasizes the significance of viscoelasticity as a crucial design parameter for biomaterials utilized in a 3D cartilage culture.

Figure 10.

(a) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining images of MSCs treated with blebbistatin or Y-27632 (day 2). Scale bars: 50 μm. (b) Representative images of type II collagen immunofluorescent staining of MSCs treated with blebbistatin or Y-27632 (day 6). Scale bars, 25 μm. Reproduced with permission from ref (73). Copyright 2023 The American Association for the Advancement of Science. (c) Representative bright-field images of hiPSC clusters on day 7 of culture in different alginate formulations with an initial elastic modulus of 20 kPa. The scale bar is 100 μm. Reproduced with permission from ref (75). Copyright 2021 WILEY-VCH. (d) Schematic representation of tetrazine-modified hyaluronan (HA) and norbornene-modified ELP creating a static covalent hydrogel (left). Schematic representation of aldehyde-modified (either benzaldehyde or aldehyde) HA and hydrazine-modified ELP creating dynamic covalent hydrogels. Dynamic slow (DYN Slow) hydrogels contain only benzaldehyde-modified HA, while dynamic fast (DYN Fast) hydrogels consist of a 1:1 mixture of benzaldehyde- and aldehyde-modified HA (right). Reproduced with permission from ref (74). Copyright 2023 The American Association for the Advancement of Science.

Beside mesenchymal stem cells, stress relaxation also has an inductive effect on neural progenitor cells (NPC). Heilshorn et al.⁷⁴ developed a viscoelastic, tunable hydrogel for studying the impact of matrix viscoelasticity on the development of NPC (Figure 10d). Initially, to create hydrogels with different stress relaxation rates, the researchers utilized hyaluronic acid (HA) modified with tetrazine and elastin-like protein (ELP) modified with norbornene to construct elastic static hydrogels (Figure 10d). Dynamic hydrogels with faster stress relaxation rates were constructed using aldehyde-modified HA and hydrazide-modified ELP, with different stress relaxation rates imparted by grafting benzaldehyde groups onto HA and pure aldehyde groups. The study found that all three hydrogels had no significant impact on NPC survival. However, compared with viscoelastic hydrogels, NPCs in purely elastic hydrogels exhibited enhanced proliferation, metabolism, and expression of the stem cell marker pHH3. TUBB3 staining indicated that NPCs in viscoelastic hydrogels exhibited better spreading and morphology closer to those of mature neurons compared to those in purely elastic hydrogels. This suggests that the stress relaxation properties of the hydrogel can regulate NPC maturation in vitro and promote differentiation into mature neurons. Chaudhuri and colleagues⁷⁵ investigated the effects of the viscoelasticity, cell-matrix adhesion ligand density, and matrix stiffness of cell growth matrix materials on the formation of lumens by human induced pluripotent stem cells (hiPSCs) (Figure 10c). The study found that increasing stress relaxation rate and RGD density promoted lumen formation, with lumen size correlating with these factors, while hydrogel hardness had no significant impact on the hiPSC viability and lumen formation.

Tumor cell behavior induction is also a key focus of research on viscoelasticity. Type 2 diabetes is a major risk factor for hepatocellular carcinoma (HCC). Increased stiffness under conditions of liver cirrhosis is known to promote the progression of HCC. Characteristics of type 2 diabetes include the accumulation of advanced glycation end products (AGEs) in the ECM. However, it is not clear how this affects HCC in nonliver cirrhosis conditions. Here, Török⁷⁶ discovered that in patients and animal models, AGEs promote changes in collagen structure and enhance the viscoelasticity of the ECM, manifested as greater viscous dissipation and faster stress relaxation, but with no change in stiffness. High levels of AGEs and viscoelasticity combined with carcinogenic β-catenin signaling promote the induction of HCC, while inhibiting AGE production and recombinant AGE clearance receptor AGER1, or disrupting AGE-mediated collagen cross-linking can reduce viscoelasticity and the growth of HCC. These results reveal that AGE-mediated structural changes enhance the viscoelasticity of the ECM, and viscoelasticity can promote cancer progression in vivo.

4.2. The Impact of Composite Viscoelastic Parameters on Cell Behavior

In contrast to single factor induced hydrogel-cell interactions, the impact of viscoelastic gels on cell behavior and fate is actually the result of the coordinated interaction of multiple parameters.⁷⁷ Recently, Mooney and co-workers⁷⁸ jointly demonstrated that the viscoelastic properties of the matrix surrounding mammary epithelial cell spheroids guide tissue proliferation in space and time. The viscoelasticity of the matrix disrupts the symmetry of the spheroids, leading to the formation of invasive finger-like protrusions, YAP nuclear translocation, and epithelial-to-mesenchymal transition, all of which depend on the Arp2/3 complex in both in vitro and in vivo settings. By computationally modeling these observed results, the researchers established a morphological stability phase diagram that correlates with matrix viscoelasticity, tissue viscosity, cellular motility, and the cell division rate. This phase diagram was validated through biochemical analyses and in vitro experiments using intestinal organoids. Furthermore, this study emphasizes the synergistic roles of viscoelasticity and stiffness in tissue growth. The growth and instability of tissues were observed in all viscoelastic gels used in these studies with the stiffness of the gels influencing the extent of these behaviors. However, in purely elastic gels, changes in stiffness had a minimal impact, as tissue growth remained slow and stable. Aiming to investigate how the viscoelastic properties of the matrix impact MSC differentiation in a 3D culture, Mooney et al.⁷⁹ developed a gel with tunable viscoelastic properties by using a single type of cross-linker, maintaining a constant concentration of alginate, and varying the molecular weights of polymers and densities of calcium ions for cross-linking. Polyethylene glycol chains were covalently linked to alginate as spacers to alter the interaction between the polymer chains. The results showed that at an initial elastic modulus of ∼9 kPa, the level of osteogenic differentiation in mesenchymal stem cells was low, with predominantly adipogenic differentiation observed. Additionally, the generation of fat decreased in the hydrogels with rapid stress relaxation. Conversely, when a higher initial elastic modulus of ∼17 kPa was used, adipogenic differentiation was not observed and osteogenic differentiation became the predominant outcome in mesenchymal stem cells. Furthermore, faster stress relaxation in the hydrogels significantly enhanced osteogenic differentiation.

Recently, Krieg group⁸⁰ reported on the fully synthetic hydrogels based on DNA libraries, which self-assembled with ultrahigh molecular weight polymers to form a dynamic DNA-cross-linked matrix (DyNAtrix). By altering DNA sequence information, DyNAtrix allows for the prediction and systematic control of its viscoelastic, thermodynamic, and kinetic parameters. Adjustable heat activation enables the uniform embedding of mammalian cells. Interestingly, the stress relaxation time can be adjusted by 4 orders of magnitude, summarizing the mechanical properties of living tissue. DyNAtrix exhibits self-healing, printability, high stability, cell and blood compatibility, and controllable degradability. Human mesenchymal stromal cells, pluripotent stem cells, canine renal cysts, and human trophoblast-like organs cultured on DyNAtrix show high vitality, proliferation, and morphogenesis.

5. Conclusion

Viscoelasticity is an inherent property of biological tissues or extracellular matrices. This mechanical property influences the behavior and fate of living cells. Investigating the mechanisms by which viscoelasticity affects cellular behavior and cell–cell interactions contributes to a deeper understanding of physiological and pathological processes, providing a foundation for tissue engineering, regenerative medicine, and related biomedical applications. In this review, we mainly explore the design and fabrication of viscoelastic hydrogels with versatile dynamic chemical bonds as well as the inductive effects of two representative viscoelastic parameters, mechanical strength and stress relaxation, on cell behaviors and fates. While merging studies on hydrogel cell-mechanics related interdisciplinary researches have been reported, several urgent issues still remain to be addressed. A primary concern is the cytotoxicity of the materials used to form the gel network. Monomers and cross-linkers that are not fully converted during synthesis can induce adverse reactions or cellular toxicity. Another issue is the degradation of the hydrogel. Over time, hydrogels degrade within the body, and the breakdown products must be nontoxic and capable of being safely metabolized or excreted. Additionally, the mechanical properties of the hydrogel must be compatible with the surrounding tissues to prevent mechanical irritation or inflammation. To address these concerns, researchers may employ several strategies: (a) Selecting biodegradable and biocompatible polymers, such as hyaluronic acid, alginate, and gelatin. (b) Optimizing synthesis methods to minimize residual monomers and cross-linkers and ensure thorough purification before application. (c) Incorporating bioactive molecules or drugs to enhance healing and reduce inflammation. (d) Rigorous testing evaluates the biocompatibility and long-term stability of hydrogels. In vitro and in vivo studies assess cytotoxicity, immune response, and degradation rates. Regulatory bodies require comprehensive safety assessments before approving hydrogels for clinical use. As for the cell-mechanics, the first issue is to precisely decouple and control the hydrogel viscoelasticity. Viscoelastic parameters usually couple each other via molecular kinetics and thermodynamics of cross-linking motifs and polymer composites, which is difficult to unravel the induction mechanism of cell behaviors of an individual viscoelastic factor. The second issue is the long-term controllability of the viscoelastic parameters. Biological tissues or extracellular matrices undergo a dynamic or homeostatic process with time-dependent degradation and regeneration. During this dynamic process, viscoelastic parameters are maintained in a relatively stable range or undergo continuous changes with the increase of age or the appearance of pathological factors. Fabrication of hydrogel biomaterials with viscoelasticity robustness or adaptive toward ever-changing physiological microenvironment is a daunting task. To maintain the long term stability and controllability of viscoelastic parameters in vivo remains an open question. The third issue is the standardization of the measurement of viscoelastic parameters. For instance, when measuring the storage modulus using rheological tests, various factors can influence the results, such as shear frequency, gap value, sample size, and parallel plate diameter. These testing conditions can significantly impact the outcome of the measurements. Furthermore, viscoelastic parameters are often measured using different equipment, such as nanoindenters, atomic force microscopes (AFMs), and rheometers. This diversity in measurement techniques leads to varied viscoelastic data, even for the same hydrogel materials and real tissue samples across different research institutions. As a result, the data are often not interoperable and lack statistical significance. Standardizing the measurement of viscoelastic parameters or developing a unified conversion method would significantly enhance the research efficiency. Standardization would involve defining consistent protocols for testing conditions and ensuring that all measurements are conducted under similar parameters. A unified conversion method would allow researchers to compare data from different instruments accurately. Such standardization and unification would facilitate better collaboration and data sharing among researchers, leading to more reliable and consistent results. It is believed that with the continuous progress in materials science, cell biology, and biomedical engineering, these issues are expected to be resolved in the future. Advancements in technology and methodologies will contribute to the development of standardized protocols and unified conversion methods. This will promote the wider and deeper application of viscoelastic hydrogels in the field of cell biology and medical science, enabling more precise and reliable studies.²⁶

The authors declare no competing financial interest.