Biomacromolecules

Collagen networks contribute to tissue architecture and modulate cellular responses in crowded three-dimensional environments. Therefore, it is the most widely used biological polymer in three-dimensional studies of cellular interactions with the extracellular matrix. In vivo, collagen exists embedded within additional matrix components. Studies have shown that the combination of matrices induces synergistic mechanical interactions, influencing the non-linear mechanical behaviour of collagen networks. However, how cells respond to changes in collagen non-linear elasticity remains largely unknown. By precisely controlling the mechanical behaviour of collagen networks with the biologically inert and semiflexible polymer polyisocyanopeptides, we demonstrate that changes in the non-linear elasticity of collagen induces morphological cell responses that influence how cells migrate, proliferate, and interact with collagen. We found that when collagen rigidifies in the presence of a second component, this induces morphological changes in cell-matrix interactions, resulting in a decrease in migration and the ability of cells to deform collagen matrices. Our results demonstrate that the onset of collagen stiffening is key to inducing intracellular tension which dictates morphological cell responses in three-dimensional collagen networks. We anticipate our findings will prove useful in understanding how cells respond to changes in collagen mechanics when combined in double network systems which better recapitulates tissues in vivo.

Hydrogels have a wide range of applications such as in biomedicine, cosmetics and soft electronics. Compared to polymer hydrogels based on covalent bonding, protein hydrogels offer distinct advantages owing to their biocompatibility and better access to molecular engineering. However, pure and natural protein hydrogels have been seldom reported except for structural proteins like collagen and silk fibrin. Here, we report the unusual ability and mechanism of a unique natural enzyme, lipoate-protein ligase A (LplA) of E. coli to self-assemble into a stimuli-responsive and reversible hydrogel of the low critical solution temperature (LCST) type. This is the first globular and catalytic protein found to form a hydrogel in response to temperature, pH and the presence of ions. Protein structure based analysis reveals the key residues responsible for the gel formation and mutational studies confirms the essential roles of hydrogen bonding between the C-terminal domains and electrostatic interactions in the N-terminal domains. Characterization of phase transitions of wild type LplA and its mutants using small angle X-ray scattering (SAXS) yields details of the gelation process from initial dimer formation over a pre-gel-state to full network development. Further electron microscopic analyses and modeling of SAXS data suggest an unusual interlinked ladder-like structure of the macroscopic crosslinking network with dimers as ladder steps. The unique features of this first reported protein hydrogel may open up hitherto inaccessible applications, especially those taking advantage of the inherent catalytic activity of LplA.

A primary objective in designing hydrogels for cell culture is recreating the cell-matrix interactions found within human tissues. Identifying the most important biomaterial features for these interactions is challenging because it is difficult to independently adjust variables such as matrix stiffness, stress relaxation, the mobility of adhesion ligands and the ability of these ligands to support cellular forces. In this work we designed a hydrogel platform consisting of interpenetrating polymer networks of covalently crosslinked poly(ethylene glycol) (PEG) and self-assembled peptide amphiphiles (PA). We can tailor the storage modulus of the hydrogel by altering the concentration and composition of each network, and we can tune the stress relaxation half-life through the non-covalent bonding in the PA network. Ligand mobility can be adjusted independently of the matrix mechanical properties by attaching the RGD cell adhesion ligand to either the covalent PEG network, the dynamic PA network, or both networks at once. Interestingly, our findings show that endothelial cell adhesion formation and spreading is maximized in soft, viscoelastic gels in which RGD adhesion ligands are present on both the covalent PEG and non-covalent PA networks. The dynamic nature of cell adhesion domains, coupled with their ability to exert substantial forces on the matrix, suggests that having different presentations of RGD ligands which are either mobile or are capable of withstanding significant forces are needed mimic different aspects of complex cell-matrix adhesions. By demonstrating how different presentations of RGD ligands affect cell behavior independently of viscoelastic properties, these results contribute to the rational design of hydrogels that facilitate desired cell-matrix interactions, with the potential of improving in vitro models and regenerative therapies.

Peptides are widely used within biomaterials to improve cell adhesion, incorporate bioactive ligands, and enable cell-mediated degradation of the matrix. While many of the peptides incorporated into biomaterials are intended to be present throughout the life of the material, their stability is not typically quantified during culture. In this work we designed a series of peptide libraries containing four different N-terminal peptide functionalizations and three C-terminal functionalization to better understand how simple modifications can be used to reduce non-specific degradation of peptides. We tested these libraries with three cell types commonly used in biomaterials research, including mesenchymal stem/stromal cells (hMSCs), endothelial cells, and macrophages, and quantified how these cell types non-specifically degraded peptide as a function of terminal amino acid and chemistry. We found that peptides in solution which contained N-terminal amines were almost entirely degraded by 48 hours, irrespective of the terminal amino acid, and that degradation occurred even at high peptide concentrations. Peptides with C-terminal carboxylic acids also had significant degradation when cultured with cells. We found that simple modifications to the termini could significantly reduce or completely abolish non-specific degradation when soluble peptides were added to cells cultured on tissue culture plastic or within hydrogel matrices, and that functionalizations which mimicked peptide conjugations to hydrogel matrices significantly slowed non-specific degradation. We also found that there were minimal differences across cell donors, and that sequences mimicking different peptides commonly-used to functionalized biomaterials all had significant non-specific degradation. Finally, we saw that there was a positive trend between RGD stability and hMSC spreading within hydrogels, indicating that improving the stability of peptides within biomaterial matrices may improve the performance of engineered matrices.

Cyclic peptides represent a burgeoning area of interest in therapeutic and biotechnological research. In opposition to their linear counterparts, cyclic peptides, such as certain ribosomally synthesized and post-translationally modified peptides (RiPPs), are more conformationally constrained and less susceptible to proteolytic degradation. The lanthipeptide RiPP cytolysin L forms a covalently enforced helical structure that may be used to disrupt helical interactions at protein-protein interfaces. Herein, an expression system is reported to produce lanthipeptides and structurally diverse cytolysin L derivatives in mammalian cells. Successful targeting of lanthipeptides to the nucleus is demonstrated. In vivo expression and targeting of such peptides in mammalian cells may allow for screening of lanthipeptide inhibitors of native protein-protein interactions. Table of contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=169 SRC="FIGDIR/small/563208v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@aafac8org.highwire.dtl.DTLVardef@13687caorg.highwire.dtl.DTLVardef@10d16caorg.highwire.dtl.DTLVardef@533398_HPS_FORMAT_FIGEXP M_FIG C_FIG

Intrinsically disordered proteins (IDPs) exhibit phase separation behavior that is closely linked to their degree of single-chain compaction, which in turn is governed by both amino acid composition and sequence patterning. Existing metrics such as sequence charge decoration (SCD) and sequence hydropathy decoration (SHD) describe these effects but are largely limited to describing differences between sequences of similar length and overall composition. In this work, we present a shuffle-based normalization scheme for SCD and SHD, enabling comparison of sequence patterning between very different IDP sequences. Leveraging this normalization scheme toward design space, we develop a Monte Carlo, based sequence design algorithm that generates novel IDPs with desired patterning features. Our design framework is further strengthened by incorporating additional metrics such as sequence aromatic decoration (SAD), compositional RMSD, and a previously developed sequence based {Delta}G predictor. We validate our approach through coarse-grained MD simulations, showing that the designed sequences exhibit tunable phase behavior. This strategy lays the groundwork for rational design of IDPs for biomedical and biotechnology applications, as well as basic biophysical research. Author summaryIntrinsically disordered proteins behave similar to polymers in solution, having no defined structure. Their behavior is dictated by the collection of shapes the protein adopts, known as its "conformational ensemble" which is tuned by its amino acid sequence, and the solution environment. In this work, we have developed parameters to describe the patterning of charged and hydrophobic amino acids within these protein sequences, which are predictive of their ability to phase separate and form dense liquid-like droplets in solution. Importantly, the parameters we develop are motivated by physics and can be applied across a large number of amino acid sequences rapidly. This will enable researchers to rapidly predict the behavior of large libraries of protein sequences. We have additionally developed a software to design randomized amino acid sequences with desired amino acid composition, and patterning properties. Finally, we have tested our design scheme and parameters by running simulations of designed IDP sequences and quantified each of their ability to phase separate.

Dynamic covalent crosslinked (DCC) hydrogels represent a significant advance in biomaterials for regenerative medicine and mechanobiology. These gels typically offer viscoelasticity and self-healing properties that more closely mimic in vivo tissue mechanics than traditional, predominantly elastic, covalent crosslinked hydrogels. Despite their promise, the effects of varying crosslinker architecture - side chain versus telechelic crosslinks - on the viscoelastic properties of DCC hydrogels have not been thoroughly investigated. This study introduces hydrazone-based alginate hydrogels and examines how side-chain and telechelic crosslinker architectures impact hydrogel viscoelasticity and stiffness. In hydrogels with side-chain crosslinking (SCX), higher polymer concentrations enhance stiffness and decelerates stress relaxation, while an off-stoichiometric hydrazine-to-aldehyde ratio leads to reduced stiffness and shorter relaxation time. In hydrogels with telechelic crosslinking, maximal stiffness and slowest stress relaxation occurs at intermediate crosslinker concentrations for both linear and star crosslinkers, with higher crosslinker valency further increasing stiffness and relaxation time. Our result suggested different ranges of stiffness and stress relaxation are accessible with the different crosslinker architectures, with SCX hydrogels leading to slower stress relaxation relative to the other architectures, and hydrogels with star crosslinking (SX) providing increased stiffness and slower stress relaxation relative to hydrogels with linear crosslinking (LX). The mechanical properties of SX hydrogels are more robust to changes induced by competing chemical reactions compared to LX hydrogels. Our research underscores the pivotal role of crosslinker architecture in defining hydrogel stiffness and viscoelasticity, providing crucial insights for the design of DCC hydrogels with tailored mechanical properties for specific biomedical applications.

Hydrogels are cross-linked polymeric networks with wide applications in drug delivery, tissue engineering, biosensing, and environmental remediation. These hydrogels additionally host living cells, small molecules and biological propagules, which further expand the applications of these materials. However, most if not all fabrication methods require covalent modifications. In this work, for the first time, we demonstrate that polymer mixtures can access an additional material state beyond the conventionally described homogeneous and two-phase regimes. By deliberately selecting polymers with a known propensity to phase separate and formulating compositions far from the binodal boundary, the system transitions directly into a mechanically stable hydrogel. We demonstrate this technique using a model system of poly (ethylene glycol) (PEG) and dextran (DEX). We have systematically characterized the hydrogels through FTIR, MALDI-TOF to discern the molecular compositions of the hydrogels. We also modulate the optical transparency of these hydrogels by varying the molecular weight of the polymers. These experimental findings are supplemented with coarse grained (CG) simulation insights to investigate the mechanistic origins of phase separation propensity with varying molecular weights of dextran. We utilized coexisting densities in the two phases using CG simulations to predict the role of dextran molecular weight on the partitioning of PEG and DEX in the two phases. Finally, we exploit the fabricated hydrogels ability to encapsulate live cells, antibiotics and plant seeds. We anticipate that this ATPS-based fabrication technique will provides a scalable, crosslinker-free route to multifunctional hydrogels enabling advanced applications in drug delivery and responsive materials.

Measuring the entropic properties of polymers such as proteins is critical to accurate prediction of their functional properties. However, the measurement of configurational entropy is possible only by low throughput techniques such as calorimetry, NMR and CD spectroscopy. Moreover, to our knowledge no system exists that allows molecular selection/enrichment based on the molecules configurational entropy. We tested the ability of the scalable tripartite GFP system to offer fine resolution of differences in configurational entropy in molecules and to isolate molecules based on their configurational entropy. The system was able to both finely resolve molecules with different configurational entropies, as well as capture them for isolation. We were able to tune the sensitivity of the system by using different mutations of the protein components. Lastly, we were able to apply the system to polypeptoid molecules and posit that the system may be applied to any other hydrophilic polymer of up to 10^3 repeating units.

Interest in utilizing amyloids to develop biomaterials is increasing due to their potential for biocompatibility, unique assembling morphology, mechanical stability, and biophysical properties. However, challenges include the complexity of peptide chemistry and the practical techniques required for processing amyloids into bulk materials. In this work, two decapeptides with fibrillar and globular morphologies were selected, blended with poly(ethylene oxide), and fabricated into composite mats via electrospinning. Notable enhancements in mechanical properties were observed, attributed to the uniform distribution of the decapeptide assemblies within the PEO matrix. Morphological differences, such as the production of thinner nanofibers, are attributed to the increased conductivity from the zwitterionic nature of the decapeptides. Blend rheology and post-processing analysis revealed how processing might affect the amyloid aggregation and secondary structure of the peptides. Both decapeptides demonstrated good biocompatibility and strong antioxidant activity, indicating their potential for safe and effective use as biomaterials. By evaluating these interdependencies, this research lays the foundation for understanding the structure-property-processing relationships of peptide-polymer blends and highlights the strong potential for developing applications in biotechnology.

The in vitro vascularization of 3D tissue constructs, such as hydrogels, remains a paramount challenge in tissue engineering. Extracellular matrix degradation and remodeling are key parts of the vascularization process; however, it is difficult to isolate the effects of degradability in both natural and synthetic matrix models. Naturally-derived matrices typically couple degradability to other material properties, whereas synthetic matrices rely on short peptide sequences to impart degradability, which typically exhibit substrate overlap to many proteases. Here, we present a method to independently and broadly tune 3D hydrogel degradation using crosslinkers with non-natural peptoid (N-substituted glycine) substitutions. Increased peptoid substitutions reduced hydrogel degradability to collagenases without altering hydrogel modulus, swelling ratio, or crosslinker length. Using this approach, human umbilical vein endothelial cells (HUVECs) encapsulated in more degradable hydrogels proliferated more, formed more vessels, exhibited higher metabolic activity, and secreted more extracellular matrix than HUVECs encapsulated in less degradable or non-degradable hydrogels. Interestingly, HUVECs encapsulated in the least degradable hydrogels secreted significantly higher matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) than HUVECs encapsulated in the most degradable hydrogels, suggesting higher MMP secretion to compensate for the reduced matrix degradability. Overall, this work highlights the importance of protease-mediated remodeling on vascularization and suggests that peptoid substitutions are effective for tuning hydrogel degradability for a variety of 3D cell applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/684851v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@c22b3corg.highwire.dtl.DTLVardef@1a4ae14org.highwire.dtl.DTLVardef@a0ad1forg.highwire.dtl.DTLVardef@c5def5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cells dynamically modify their local extracellular matrix by expressing proteases that degrade matrix proteins. This enables cells to spread and migrate within tissues, and this process is often mimicked in hydrogels through the incorporation of peptide crosslinks that can be degraded by cell-secreted proteases. However, the cleavage of hydrogel crosslinks will also reduce the local matrix mechanical properties, and most crosslinking peptides, such as the widely used GPQGIWGQ "PanMMP" sequence, lead to bulk degradation of the hydrogel. A subset of proteases are localized to the cell membrane and are only active in the pericellular region in the immediate vicinity of the cell surface. These membrane-type proteases have important physiological roles and enable cells to migrate within tissues. In this work we developed an approach to identify and optimize peptide sequences that are specifically degraded by membrane-type proteases. We utilized a proteomic screen to identify peptide targets, and coupled this with a functional assay that both quantifies peptide degradation by individual cell types and can elucidate whether the peptides are primarily cleaved by soluble proteases or membrane-type proteases. We then used a split-and-pool synthesis approach to generate more than 300 variants of the target peptide to improve the degradation behavior. We identified an optimized peptide sequence, KLVADLMASAE, which is primarily degraded by membrane-type proteases, but enables both endothelial cells and stem cells grown in KLVADLMASAE-crosslinked hydrogels to spread and have viabilities similar to the gels crosslinked by the PanMMP peptide. Notably, the biological performance of the KLVADLMASAE peptide-cross linked gels was significantly improved from the initial peptide target found in the proteomic screen. This work introduces a functional approach to identifying and refining protease-substrate peptides as a way to enhance the properties of hydrogel matrices.

Proteinaceous amyloids are well known for their widespread pathological roles but lately have emerged also as key components in several biological functions. The remarkable ability of amyloid fibers to form tightly packed conformations in a cross {beta}-sheet arrangement manifests in their robust enzymatic and structural stabilities. These characteristics of amyloids make them attractive for designing proteinaceous biomaterials for various biomedical and pharmaceutical applications. In order to design customizable and tunable amyloid nanomaterials, it is imperative to understand the sensitivity of the peptide sequence for subtle changes based on amino acid position and chemistry. Here we report our results from four rationally-designed amyloidogenic decapeptides that subtly differ in hydrophobicity and polarity at positions 5 and 6. We show that making the two positions hydrophobic renders the peptide with enhanced aggregation and material properties while the introduction of polar residues in position 5 dramatically changes the structure and nanomechanical properties of the fibrils formed. A charged residue at position 6, however, completely abrogates amyloid formation. In sum, we show that subtle changes in the sequence do not make the peptide innocuous but rather sensitive to aggregation, reflected in the biophysical and nanomechanical properties of the fibrils. We conclude that tolerance of peptide amyloid for subtle changes in the sequence should not be neglected for the effective design of customizable amyloid nanomaterials.

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size [~]7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

Thiol-ene click chemistry is a powerful tool for designing hydrogels mimicking the mechanical and biochemical properties of 3D cellular microenvironments. The high selectivity of thiol-norbornene step-growth polymerization enables precise control of crosslinking mechanism, circumventing the alkene homopolymerization present in other systems that can prevent encapsulated cell spreading. Limited stress relaxation of a dynamically-crosslinked norbornene-modified hyaluronic acid (NorHA) hydrogel employing a thiol-norbornene photoclick reaction led us to investigate the prevalence of norbornene homopolymerization in this supposed click reaction. Norbornene conversion was quantified in multiple thiol plus norbornene-modified polymer system permutations, revealing higher norbornene conversion than expected for 1:1 thiol-ene addition. We showed that decreasing the number of norbornenes per NorHA chain (f) mitigated network formation via norbornene homopolymerization. Dynamic hydrogels fabricated with NorHA of f = 8 (Nor8HA) exhibited 93.0 {+/-} 1.6% relaxation, while those fabricated with NorHA of f = 40 (Nor40HA) achieved only 42.3 {+/-} 0.1% relaxation. As early as day 3 of culture, Nor8HA hydrogels facilitated spreading of encapsulated human mesenchymal stromal cells (hMSCs) into a spindle-like morphology (aspect ratio: 2.95 {+/-} 0.38), while Nor40HA hydrogels appeared to constrain cells into a spherical or compact star morphology (aspect ratio: 1.22 {+/-} 0.01). Inference of a single-cell morphological space derived from a shape-matching distance metric validated the two distinct hMSC morphological phenotypes primarily associated with polymer f. Despite its widespread use as a click reaction, radical-mediated thiol-norbornene crosslinking was found to not be stoichiometric in dilute aqueous conditions used to fabricate hydrogels. Altering network topology through polymer f enabled the rescue of hydrogel dynamic behavior and encapsulated hMSC spreading, despite the presence of norbornene homopolymerization, highlighting the need to consider network-level properties when designing engineered cellular microenvironments.

Protein liquid-liquid phase separation underlies the formation of membraneless organelles in cells and performs a key role in the assembly process of natural materials such as the assembly of tropoelastin into elastic fibers. Here, we engineered a series of charged elastin-like polypeptides (ELPs) that form complex coacervates, providing a rapid method to concentrate proteins into a fluid state. Compared to coacervates formed from simple coacervation, complex coacervates exhibited greater fluidity, likely due to differences between electrostatic interactions and hydrophobic forces. We designed these ELPs to further contain crosslinking domains compatible with tyrosinase or transglutaminase and found that crosslinking was enhanced when proteins were in a complex coacervate compared to free in solution. Crosslinking the ELP complex coacervates led to the formation of gels with distinct properties dependent on the nature of the crosslinking. This work expands the design space of ELP hydrogels, offering a novel strategy for forming crosslinked networks from complex coacervates and providing opportunity for future use in tissue engineering and biocompatible biomaterials applications.

The glycosaminoglycan hyaluronan (HA) is an essential and ubiquitous component of human tissues and biofluids. The only known covalent modification of HA entails the attachment of heavy chains (HC) from the inter-alpha-inhibitor (II) family of proteoglycans, forming stable complexes (HC*HA) that arise during inflammation. In some contexts, HC*HA is thought to contribute to pathology, whereas in others it may form part of a protective pathway. However, its exact roles are not fully understood. Here, we report that HC modifications can protect HA from fragmentation by reactive oxygen species (ROS) produced during the inflammatory cascade. Using solid-state nanopore molecular size analysis, we show that HA is highly resistant to degradation from exogenous ROS in vitro when in the context of HC*HA complexes, while the unmodified HA polymer is fragmented rapidly under the same conditions. Experiments performed with admixtures of HA and unbound antioxidant proteins - including HC-bearing components - demonstrate the necessity of covalent HC attachment to the polysaccharide for the protection. Finally, we find that HA with high-HC content from inflammatory equine synovial fluid has increased resilience to ROS damage compared to low-HC HA from a healthy joint. Collectively, these results demonstrate that covalent HC modification is an effective biological strategy for preserving HA integrity against ROS fragmentation, including in inflammatory conditions.

The formation of interpenetrating polymer networks (IPNs) in polysaccharide-based hydrogels is influenced by ion valency and coordination chemistry. In this work, hyaluronic acid (HA) and kappa-carrageenan ({kappa}-CG) were combined at low polymer concentrations suitable for injectable hydrogel applications and enriched with monovalent (K+), divalent (Ca2+), and trivalent (Al3+) ions to investigate ion-specific contributions to network formation, structure and recovery. FTIR spectroscopy showed that K+ did not produce detectable sulfate or carboxylate shifts, consistent with a predominantly physical mixture and the absence of IPN formation. Ca2+ induced concentration-dependent shifts in both the amide/carboxylate region and the sulfate band (1232 [->] 1236 cm-1), absent at a low concentration but reemerging at higher concentrations, consistent with local chain compaction, complexation of HA carboxylates and bridging between{kappa} -CG helices, indicating promotion of a semi-interpenetrated network structure above a thresh-old concentration. Al3+ induced a distinct shoulder in the HA carboxylate region, confirming HA coordination and co-crosslinking with{kappa} -CG, yielding a semi-IPN. Microrheology showed progressively stronger local confinement with increasing ion valency corroborating insights from spectroscopy, while recovery tests showed Ca2+ attained the highest recovery potential possibly due to more robust network formation, Al3+ systems displayed moderate recovery but overscreening at high concentrations resulted in network collapse, and K+ systems displayed poor recovery.Importantly, these ion-specific outcomes must be interpreted in the context of combined electrostatic screening effects, which contribute to charge neutralization and polymer chain association independent of coordination chemistry. Collectively, these findings highlight ion charge as critical design levers which may be leveraged in tailoring mechanical properties of interpenetrating network hydrogels for injectable applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/675661v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1924ee2org.highwire.dtl.DTLVardef@7916cforg.highwire.dtl.DTLVardef@1c89221org.highwire.dtl.DTLVardef@f1c2c7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIncorporation of{kappa} -carrageenan ({kappa}CG) into a chemically unmodified hyaluronic-acid (HA) based hydrogel even at low concentrations is a potentially viable route for more efficient structuring. C_LIO_LIInterpenetrating network formation, structure and recovery of HA-{kappa} CG networks can be tuned by charge density and coordination behavior of the crosslinking ion. C_LIO_LICa2+ reinforces{kappa} CG junction zones, yielding the highest elastic moduli and recovery capacity, with evidence of semi-IPN formation pointing to more robust network formation relative to Al3+ and K+. C_LI

Efforts to model and repair connective tissue through engineered tissue constructs have generated great interest in culturing cells in 3d polymer network environments. It has been shown that the polymer environment is influential in determining cellular responses such as differentiation, migration and morphology. Hydrogels are used to mimic the cellular microenvironment, but in most cases hydrogels consisting of one polymeric component are used whereas tissues are composites of different polymers. A clear understanding of how different extracellular components and their mechanical characteristics influence cell behaviour is lacking. Here we developed and characterised composite hydrogels of hyaluronan and fibrin and evaluated their use for cartilage tissue engineering. We demonstrate that these cartilage-mimicking composites have a higher stiffness relative to the individual constituents. Next, we cultured human mesenchymal stromal cells in these 3D hydrogels with chondrogenic media and revealed marked differences in cell morphology, gene expression and cartilage-like matrix deposition depending on the specific extracellular composition. We found that, despite evidence for strong adhesion of the cells to fibrin networks in 2D systems, in 3D systems the primary determinant of cellular morphology is the significantly denser hyaluronan network. Dense hyaluronan hydrogels cause local cell confinement evidenced by rounder cell morphologies, independent of the presence of fibrin. While the composite fibrin-hyaluronan hydrogels led to lower expression of chondrogenic genes than hyaluronan alone, the larger linear modulus and resistance to cell-mediated contraction due to the composite nature of the matrix provides a strong advantage in terms of macroscopic mechanical stability. These findings highlight the potential of multi-component hydrogels for controlling cellular behaviour and bulk mechanical properties of cell-hydrogel constructs independently, therefore opening avenues for better understanding the complex interplay between cells and their extracellular environment and thus improve the biofabrication of connective tissues for disease modelling and tissue regeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/555478v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@151962org.highwire.dtl.DTLVardef@1358890org.highwire.dtl.DTLVardef@198dcedorg.highwire.dtl.DTLVardef@d04f80_HPS_FORMAT_FIGEXP M_FIG C_FIG

Phase separation plays an important role in the formation of membraneless compartments within the cell, and intrinsically disordered proteins with low-complexity sequences can drive this compartmentalisation. Various intermolecular forces, such as aromatic-aromatic and cation-aromatic interactions, promote phase separation. However, little is known about how the ability of proteins to phase separate under physiological conditions is encoded in their energy landscapes, and this is the focus of the present investigation. Our results provide a first glimpse into how the energy landscapes of minimal peptides that contain{pi} -{pi} and cation-{pi} interactions differ from the peptides that lack amino acids with such interactions. The peaks in the heat capacity (CV) as a function of temperature report on alternative low-lying conformations that differ significantly in terms of their enthalpic and entropic contributions. The CV analysis and subsequent quantification of frustration of the energy landscape suggest that the interactions that promote phase separation leads to features (peaks or inflection points) at low temperatures in CV, more features may occur for peptides containing residues with better phase separation propensity and the energy landscape is more frustrated for such peptides. Overall, this work links the features in the underlying single-molecule potential energy landscapes to their collective phase separation behaviour, and identifies quantities (CV and frustration metric) that can be utilised in soft material design.