Native chemical ligation (NCL) offers a route to form amide-linked hydrogels under mild aqueous conditions, yet its implementation in biomaterials has been constrained by slow kinetics, free-thiol byproducts, and inhibition in chemically complex environments. Here, we demonstrate that deliberate electrophile design through incorporation of a strain encoded βthiolactone enables rapid and selective NCL-mediated hydrogel formation with a “kinetic proofreading” mechanism. A penicillamine-derived β-thiolactone cross-linker synthesized directly on four-arm polyethylene glycol (PEG, 10 kDa) exhibits fast gelation (tgel ≈ 108 s) in complete cell culture media while maintaining orthogonality to embedded human dermal fibroblasts. Relative to a conventional alkyl thioester and a γ-thiolactone analogue, the strained β-thiolactone displays accelerated gelation and enhanced tolerance to competing endogenous thiols. Mechanistically, geminal dimethyl substitution promotes rapid β-thiolactone recyclization, suppressing unproductive thiol exchange while productive NCL proceeds through an irreversible S-to-N acyl shift. This kinetic control enables direct peptide incorporation during gelation, hydrogel microfiber fabrication, and long-term three-dimensional cell encapsulation. These findings establish electrophile strain and recyclization kinetics as design parameters for engineering selective and mechanically functional covalent biomaterials.

A central challenge in biomaterials design is developing cross-linking reactions that are fast, selective, synthetically accessible, and compatible with the nucleophile-rich environments required for cell encapsulation. Native chemical ligation (NCL) offers an attractive route to amide-linked hydrogels under mild aqueous conditions, yet its implementation in biomaterials has been constrained by slow kinetics, free-thiol byproducts, and inhibition in complex media. Here, we demonstrate that deliberate electrophile design through incorporation of a strain-encoded β-thiolactone enables rapid and selective NCL-mediated hydrogel formation through rapid recyclization of off-target intermediates. A penicillamine-derived β-thiolactone cross-linker synthesized directly on four-arm polyethylene glycol (PEG, 10 kDa) exhibits fast gelation in complete cell culture media while maintaining orthogonality to embedded human dermal fibroblasts. Relative to a conventional alkyl thioester and a γ-thiolactone analogue, the strained β-thiolactone displays accelerated gelation and enhanced tolerance to competing endogenous thiols. Mechanistically, geminal dimethyl substitution promotes rapid β-thiolactone recyclization, suppressing unproductive thiol exchange while productive NCL proceeds through an irreversible S-to-N acyl shift. Because unreacted β-thiolactones persist under physiological conditions, the network remains chemically addressable after gelation, enabling temporally delayed functionalization with N-Cys-containing molecules. This combination of rapid network formation and postgelation addressability enables direct peptide incorporation, hydrogel microfiber fabrication, and long-term three-dimensional cell encapsulation.

Understanding polymer-surfactant interactions is essential for regulating phase transition and polymer aggregation, enabling the design of functional materials with tailored properties. Here, we introduce programmable dextran-based thermoresponsive polysaccharide condensates that exhibit reversible phase transitions with tunable lower critical solution temperatures. Photo-initiated radical polymerization permits hydrogel crosslinking, harnessing phase separation to generate hydrogels with distinct microstructures and mechanical heterogeneity. We systematically investigate the impact of anionic sodium dodecyl sulfate (SDS), cationic hexadecyltrimethylammonium bromide (CTAB), nonionic Pluronic F-127, and zwitterionic 3-[(3-cholamidopropyl) dimethylammonio]−1-propanesulfonate (CHAPS) surfactants on phase transition dynamics. Surfactant charge density, hydrophilic-lipophilic balance (HLB), and critical micelle concentrations (CMC) collectively govern temperature-triggered phase separation. The resulting photo-crosslinked gels demonstrate surfactant-specific microstructures, including core-shell domains, interconnected elongated micelles, and dual emulsions. Micromechanical characterization exhibits structurally coordinated stiffness and adhesion, where Pluronic forms core-shell structures with reduced adhesion, while CTAB presents elongated structures and lowered modulus. These findings provide a framework for tailoring surfactant-polysaccharide interactions to direct microstructure-property-performance relationships in biocomposite materials design. This paper reports programmable dextran-based thermoresponsive polysaccharide hydrogels that exhibit reversible phase transitions with tunable lower critical solution temperatures.

A major challenge in tissue engineering involves the development of synthetic biomaterials that effectively induce and maintain functional vascularization of engineered tissue constructs post implantation. While conjugating heparin to a dextran hydrogel developed a pro-angiogenic scaffold that led to substantial endothelial multicellular assembly in vitro and enhanced host vessel invasion in vivo, the inherent anti-coagulant bioactivities of native heparin elicited substantial local bleeding upon implantation. To decouple the pro-angiogenic effects from the anti-coagulant activity, we developed a synthetic, heparin-mimetic material by introducing sulfate adducts to the dextran backbone. These heparin-mimetic hydrogels bound and immobilized growth factors, enhanced angiogenic signaling, and promoted both in vitro vascular network formation in 3D and in vivo tissue microvascularization to a similar extent as heparin conjugated hydrogels, but without inducing local bleeding at implantation sites. This development of a fully synthetic, highly tunable angiogenic biomaterial provides a new material system to engineer functional vascularized tissues.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.

While the extracellular matrix (ECM) has long been recognized for its structural contributions, anchoring cells for adhesion, providing mechanical support, and maintaining tissue integrity, recent efforts have elucidated its dynamic, reciprocal, and diverse properties on angiogenesis. The ECM modulates angiogenic signaling and mechanical transduction, influences the extent and degree of receptor activation, controls cellular behaviors, and serves as a reservoir for bioactive macromolecules. Collectively, these factors guide the formation, maturation, and stabilization of a functional vascular network. This review aims to shed light on the versatile roles of the ECM in angiogenesis, transcending its traditional functions as a mere structural material. We will explore its engagement and synergy in signaling modulation, interactions with various angiogenic factors, and highlight its importance in both health and disease. By capturing the essence of the ECM's diverse functionalities, we highlight the significance in the broader context of vascular biology, enabling the design of novel biomaterials to engineer vascularized tissues and their potential therapeutic implications.

In brain metastasis, cancer cells remain in close contact with the existing vasculature and can use vessels as migratory paths—a process known as vessel co-option. However, the mechanisms regulating this form of migration are poorly understood. Here we use ex vivo brain slices and an organotypic in vitro model for vessel co-option to show that cancer cell invasion along brain vasculature is driven by the difference in stiffness between vessels and the brain parenchyma. Imaging analysis indicated that cells move along the basal surface of vessels by adhering to the basement membrane extracellular matrix. We further show that vessel co-option is enhanced by both the stiffness of brain vasculature, which reinforces focal adhesions through a talin-dependent mechanism, and the softness of the surrounding environment that permits cellular movement. Our work reveals a mechanosensing mechanism that guides cell migration in response to the tissue’s intrinsic mechanical heterogeneity, with implications in cancer invasion and metastasis. Uroz et al. report that the distinct mechanical properties of brain vasculature versus parenchyma drive cancer cell migration through a talin-dependent mechanism, enabling vessel co-option and metastatic invasion in the brain.

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall, diastolic dysfunction, and fibrosis, and is associated with mutations in genes encoding sarcomere proteins. While in vitro studies have used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to study HCM, these models have not examined the multicellular interactions involved in fibrosis. Using engineered cardiac microtissues (CMTs) composed of HCM-causing MYH7 -variant hiPSC-CMs and wild-type fibroblasts, we observed cell-cell cross-talk leading to increased collagen deposition, tissue stiffening, and decreased contractility dependent on fibroblast proliferation. hiPSC-CM conditioned media and single-nucleus RNA sequencing data suggested that fibroblast proliferation is mediated by paracrine signals from MYH7 -variant cardiomyocytes. Furthermore, inhibiting epidermal growth factor receptor tyrosine kinase with erlotinib hydrochloride attenuated stromal activation. Last, HCM-causing MYBPC3 -variant CMTs also demonstrated increased stromal activation and reduced contractility, but with distinct characteristics. Together, these findings establish a paracrine-mediated cross-talk potentially responsible for fibrotic changes observed in HCM.

3D cancer spheroids are vital in vitro models for cancer drug discovery. The current cancer spheroid generation utilizes low-adhesion plates or forced aggregation of single cells. However, certain cancers do not form stable spheroids in these protocols, nor do pre-formed tumor spheroids recapitulate in vivo development and progression. This project aims to overcome this limitation with the use of a biomimetic hydrogel system to mimic in vivo cancer cell growth and tumor development and use it as an in vitro platform for 3D cancer drug screening. The biocompatible and tunable hydrogel permits independent control of key biochemical and biophysical cues such as matrix degradation, cell adhesion, and stiffness; leading to systematic investigation of matrix parameter contribution to regulating in vitro tumor growth and treatment response. We modified a homo-polysaccharide dextran with methacrylate groups and generated dextran macromers (Dex-MA) containing ample reactive methacrylates per polymer. Cell adhesion is mediated by thiol-terminated RGD peptide added to the methacrylates, followed by crosslinking with di-thiolated MMP-labile peptides through Michael-type addition reaction. Varying the sequence of the crosslinker furthermore gives access to a range of degradability, independent of matrix stiffness. Our experiments showed that Dex-MA supported robust growth of SK-BR-3 breast cancer cells from single cell to 3D tumor spheroids, whereas the cell line failed to form tumor aggregates in low-adhesion plates. Alteration of hydrogel stiffness further revealed stiffness dependence of tumor growth, invasion, and response to treatment, suggesting the importance of matrix properties on regulating cell behavior. This dextran-based tunable hydrogel system provides key insights into how engineered matrix properties control tumor growth, critical for screening novel therapy compounds, and may reveal mechanisms for 3D tumor development, invasion, metastasis, and drug resistance. Citation Format: Jaxson R. Libby, Linqing Li, Sarah R. Walker. Matrix properties regulate 3D tumor spheroid growth and cancer drug response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4556.

Thermoresponsive polysaccharide-based materials with tunable transition temperatures regulating phase-separated microdomains offer substantial opportunities in tissue engineering and biomedical applications. To develop novel synthetic thermoresponsive polysaccharides, we employed versatile chemical routes to attach hydrophobic adducts to the backbone of hydrophilic dextran and gradually increased the hydrophobicity of the dextran chains to engineer phase separation. Conjugating methacrylate moieties to the dextran backbone yielded a continuous increase in macromolecular hydrophobicity that induced a reversible phase transition whose lower critical solution temperature can be modulated via variations in polysaccharide concentration, molecular weight, degree of methacrylation, ionic strength, surfactant, urea and Hofmeister salts. The phase separation is driven by increased hydrophobic interactions of methacrylate residues, where the addition of surfactant and urea disassociates hydrophobic interactions and eliminates phase transition. Morphological characterization of phase-separated dextran solutions via scanning electron and flow imaging microscopy revealed the formation of microdomains upon phase transition. These novel thermoresponsive dextrans exhibited promising cytocompatibility in cell culture where the phase transition exerted negligible effects on the attachment, spreading and proliferation of human dermal fibroblasts. Leveraging the conjugated methacrylate groups, we employed photo-initiated radical polymerization to generate phase-separated hydrogels with distinct microdomains. Our bottom-up approach to engineering macromolecular hydrophobicity of conventional hydrophilic, non-phase separating dextrans to induce robust phase transition and generate thermoresponsive phase-separated biomaterials will find applications in mechanobiology, tissue repair and regenerative medicine.

Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch-dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility 1 – 3 . Although all of these processes consume energy 4 , 5 , it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.

Cell-generated tractions play an important role in various physiological and pathological processes such as stem-cell differentiation, cell migration, wound healing, and cancer metastasis. Traction force microscopy (TFM) is a technique for quantifying cellular tractions during cell-matrix interactions. Most applications of this technique have heretofore assumed that the matrix surrounding the cells is linear elastic and undergoes infinitesimal strains, but recent experiments have shown that the traction-induced strains can be large (e.g., more than 50%). In this paper, we propose a novel three-dimensional (3D) TFM approach that consistently accounts for both the geometric nonlinearity introduced by large strains in the matrix, and the material nonlinearity due to strain-stiffening of the matrix. In particular, we pose the TFM problem as a nonlinear inverse hyperelasticity problem in the stressed configuration of the matrix, with the objective of determining the cellular tractions that are consistent with the measured displacement field in the matrix. We formulate the inverse problem as a constrained minimization problem and develop an efficient adjoint-based minimization procedure to solve it. We first validate our approach using simulated data, and quantify its sensitivity to noise. We then employ the new approach to recover tractions exerted by NIH 3T3 cells fully encapsulated in hydrogel matrices of varying stiffness. We find that neglecting nonlinear effects can induce significant errors in traction reconstructions. We also find that cellular tractions roughly increase with gel stiffness, while the strain energy appears to saturate.

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by coculturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly can be useful for clinical translation. In this study, a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis) is introduced, whereby fibroblasts are selectively ablated on demand, and it is utilized to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function is demonstrated. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering‐via‐elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.

Vocal fold scar, characterized by alterations in the lamina propria extracellular matrix, disrupts normal voice quality and function. Due to a lack of satisfactory clinical treatments, there is a need for tissue engineering strategies to restore voice. Candidate biomaterials for vocal fold tissue engineering must match the unique biomechanical and viscoelastic properties of native tissue without provoking inflammation. We sought to introduce elastomeric properties to hyaluronic acid (HA)-based biomaterials by incorporating resilin-like polypeptide (RLP) into hybrid hydrogels. Physically crosslinked RLP/HA and chemically crosslinked RLP-acrylamide/thiolated HA (RLP-AM/HA-SH) hydrogels were fabricated using cytocompatible chemistries. Mechanical properties of hydrogels were assessed in vitro using oscillatory rheology. Hybrid hydrogels were injected into rabbit vocal folds and tissues were assessed using rheology and histology. A small number of animals underwent acute vocal fold injury followed by injection of RLP-AM/HA-SH hydrogel alone or as a carrier for human bone marrow mesenchymal stem cells (BM-MSCs). Rheological testing confirmed that mechanical properties of materials in vitro resembled native vocal fold tissue and that viscoelasticity of vocal fold mucosa was preserved days 5 and 21 after injection. Histological analysis revealed that hybrid hydrogels provoked only mild inflammation in vocal fold lamina propria with demonstrated safety in the airway for up to 3 weeks, confirming acute biocompatibility of crosslinking chemistries. After acute injury, RLP-AM/HA-SH gel with and without BM-MSCs did not result in adverse effects or increased inflammation. Collectively, results indicate that RLP and HA-based hybrid hydrogels are highly promising for engineering the vocal fold lamina propria. Lay Summary To date, there are no satisfactory treatments for vocal fold scar, which has significant negative impact on communication and quality of life. Tissue engineering holds promise for restoring stiff connective tissue in scarred vocal folds to normal pliability and flexibility. Here, we synthesized and characterized hybrid hydrogels that combine hyaluronic acid (HA), a component of native vocal fold tissue, with resilin-like polypeptide (RLP), a highly elastic and resilient biomaterial. Viscoelasticity and biocompatibility were assessed after injection in rabbit vocal folds. Hybrid hydrogels mimicked mechanical properties of normal vocal folds in vitro and in vivo with minimal inflammation in the airway. RLP/HA hybrid hydrogels thus represent a potential biomaterial to restore normal vocal fold vibration and improve voice quality and function.

Heterogeneous hydrogels with desired matrix complexity are studied for a variety of biomimetic materials. Despite the range of such microstructured materials described, few methods permit independent control over microstructure and microscale mechanics by precisely controlled, single-step processing methods. Here, a phototriggered crosslinking methodology that traps microstructures in liquid-liquid phase-separated solutions of a highly elastomeric resilin-like polypeptide (RLP) and poly(ethylene glycol) (PEG) is reported. RLP-rich domains of various diameters can be trapped in a PEG continuous phase, with the kinetics of domain maturation dependent on the degree of acrylation. The chemical composition of both hydrogel phases over time is assessed via in situ hyperspectral coherent Raman microscopy, with equilibrium concentrations consistent with the compositions derived from NMR-measured coexistence curves. Atomic force microscopy reveals that the local mechanical properties of the two phases evolve over time, even as the bulk modulus of the material remains constant, showing that the strategy permits control of mechanical properties on micrometer length scales, of relevance in generating mechanically robust materials for a range of applications. As one example, the successful encapsulation, localization, and survival of primary cells are demonstrated and suggest the potential application of phase-separated RLP-PEG hydrogels in regenerative medicine applications.

Vocal folds are connective tissues housed in the larynx, which can be subjected to various injuries and traumatic stimuli that lead to aberrant tissue structural alterations and fibrotic-induced biomechanical stiffening observed in patients with voice disorders. Much effort has been devoted to generate soft biomaterials that are injectable directly to sites of injury. To date, materials applied toward these applications have been largely focused on natural extracellular matrix-derived materials such as collagen, fibrin or hyaluronic acid; these approaches have suffered from the fact that materials are not sufficiently robust mechanically nor offer sufficient flexibility to modulate material properties for targeted injection. We have recently developed multiple resilin-inspired elastomeric hydrogels that possess similar mechanical properties as those reported for vocal fold tissues, and that also show promising in vitro cytocompatibility and in vivo biocompatibility. Here we report studies that test the delivery of resilin-based hydrogels through injection to the subcutaneous tissue in a wild-type mice model; histological and genetic expression outcomes were monitored. The rapid kinetics of crosslinking enabled facile injection and ensured the rapid transition of the viscous resilin precursor solution to a solid-like hydrogel in the subcutaneous space in vivo; the materials exhibited storage shear moduli in the range of 1000-2000 Pa when characterized through oscillatory rheology. Histological staining and gene expression profiles suggested minimal inflammatory profiles three weeks after injection, thereby demonstrating the potential suitability for site-specific in vivo injection of these elastomeric materials. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2229-2242, 2018.

Detailed understanding of the local structure-property relationships in soft biopolymeric hydrogels can be instrumental for applications in regenerative tissue engineering. Resilin-like polypeptide (RLP) hydrogels have been previously demonstrated as useful biomaterials with a unique combination of low elastic moduli, excellent resilience, and cell-adhesive properties. However, comprehensive mechanical characterization of RLP hydrogels under both low-strain and high-strain conditions has not yet been conducted, despite the unique information such measurements can provide about the local structure and macromolecular behavior underpinning mechanical properties. In this study, mechanical properties (elastic modulus, resilience, and fracture initiation toughness) of equilibrium swollen resilin-based hydrogels were characterized via oscillatory shear rheology, small-strain microindentation, and large-strain puncture tests as a function of polypeptide concentration. These methods allowed characterization, for the first time, of the resilience and failure in hydrogels with low polypeptide concentrations (<20 wt%), as the employed methods obviate the handling difficulties inherent in the characterization of such soft materials via standard mechanical techniques, allowing characterization without any special sample preparation and requiring minimal volumes (as low as 50 μL). Elastic moduli measured from small-strain microindentation showed good correlation with elastic storage moduli obtained from oscillatory shear rheology at a comparable applied strain rate, and evaluation of multiple loading-unloading cycles revealed decreased resilience values at lower hydrogel concentrations. In addition, large-strain indentation-to-failure (or puncture) tests were performed to measure large-strain mechanical response and fracture toughness on length scales similar to biological cells (∼10-50 μm) at various polypeptide concentrations, indicating very high fracture initiation toughness for high-concentration hydrogels. Our results establish the utility of employing microscale mechanical methods for the characterization of the local mechanical properties of biopolymeric hydrogels of low concentrations (<20 wt%), and show how the combination of small and large-strain measurements can provide unique insight into structure-property relationships for biopolymeric elastomers. Overall, this study provides new insight into the effects on local mechanical properties of polypeptide concentration near the overlap polymer concentration c* for resilin-based hydrogels, confirming their unique elastomeric features for applications in regenerative medicine.