We study the relation between the macroscopic viscoelastic properties of aqueous hyaluronan polymer solutions and the molecular-scale dynamics of water using rheology measurements, differential dynamic microscopy, and polarization-resolved infrared pump-probe spectroscopy. We observe that the addition of hyaluronan to water leads to a slowing down of the reorientation of a fraction of the water molecules. Near pH 2.4, the viscosity of the hyaluronan solution reaches a maximum, while the number of slowed down water molecules reaches a minimum. This implies that the water molecules become on average more mobile when the solution becomes more viscous. This observation indicates that the increase in viscosity involves the expulsion of hydration water from the surfaces of the hyaluronan polymers, and a bundling of the hyaluronan polymer chains.

The formation and destabilization of viscoelastic filaments are of importance in many industrial and biological processes. Filament instabilities have been observed for viscoelastic fluids but recently also for soft elastic solids. In this work, we address the central question of how to connect the dynamical behavior of viscoelastic liquids to that of soft elastic solids. We take advantage of a biopolymer material whose viscoelastic properties can be tuned over a very large range by its pH, and study the destabilization and ensuing instabilities in uniaxial extensional deformation. In agreement with very recent theory, we find that the interface shapes dictated by the instabilities converge to an identical similarity solution for low-viscosity viscoelastic fluids and highly elastic gels. We thereby bridge the gap between very fluid and strongly elastic materials. In addition, we provide direct evidence that at late times an additional filament instability occurs due to a dynamical phase separation.

Fibrin is an elastomeric protein forming highly extensible fiber networks that provide the scaffold of blood clots. Here we reveal the molecular mechanisms that explain the large extensibility of fibrin networks by performing in situ small angle X-ray scattering measurements while applying a shear deformation. We simultaneously measure shear-induced alignment of the fibers and changes in their axially ordered molecular packing structure. We show that fibrin networks exhibit distinct structural responses that set in consecutively as the shear strain is increased. They exhibit an entropic response at small strains (<5%), followed by progressive fiber alignment (>25% strain) and finally changes in the fiber packing structure at high strain (>100%). Stretching reduces the fiber packing order and slightly increases the axial periodicity, indicative of molecular unfolding. However, the axial periodicity changes only by 0.7%, much less than the 80% length increase of the fibers, suggesting that fiber elongation mainly stems from uncoiling of the natively disordered αC-peptide linkers that laterally bond the molecules. Upon removal of the load, the network structure returns to the original isotropic state, but the fiber structure becomes more ordered and adopts a smaller packing periodicity compared to the original state. We conclude that the hierarchical packing structure of fibrin fibers, with built-in disorder, makes the fibers extensible and allows for mechanical annealing. Our results provide a basis for interpreting the molecular basis of haemostatic and thrombotic disorders associated with clotting and provide inspiration to design resilient bio-mimicking materials. Statement of Significance: Fibrin provides structural integrity to blood clots and is also widely used as a scaffold for tissue engineering. To fulfill their biological functions, fibrin networks have to be simultaneously compliant like skin and resilient against rupture. Here, we unravel the structural origin underlying this remarkable mechanical behaviour. To this end, we performed in situ measurements of fibrin structure across multiple length scales by combining X-ray scattering with shear rheology. Our findings show that fibrin sustains large strains by undergoing a sequence of structural changes on different scales with increasing strain levels. This demonstrates new mechanistic aspects of an important biomaterial's structure and its mechanical function, and serves as an example in the design of biomimicking materials.

Collagen forms the structural scaffold of connective tissues in all mammals. Tissues are remarkably resistant against mechanical deformations because collagen molecules hierarchically self-assemble in fibrous networks that stiffen with increasing strain. Nevertheless, collagen networks do fracture when tissues are overloaded or subject to pathological conditions such as aneurysms. Prior studies of the role of collagen in tissue fracture have mainly focused on tendons, which contain highly aligned bundles of collagen. By contrast, little is known about fracture of the orientationally more disordered collagen networks present in many other tissues such as skin and cartilage. Here, we combine shear rheology of reconstituted collagen networks with computer simulations to investigate the primary determinants of fracture in disordered collagen networks. We show that the fracture strain is controlled by the coordination number of the network junctions, with less connected networks fracturing at larger strains. The hierarchical structure of collagen fine-tunes the fracture strain by providing structural plasticity at the network and fiber level. Our findings imply that low connectivity and plasticity provide protective mechanisms against network fracture that can optimize the strength of biological tissues.

Many biopolymer hydrogels are environmentally responsive because they are held together by physical associations that depend on pH and temperature. Here, we investigate how the pH and temperature responses of the rheology of hyaluronan hydrogels are connected to the underlying molecular interactions. Hyaluronan is an essential structural biopolymer in the human body with many applications in biomedicine. Using two-dimensional infrared spectroscopy, we show that hyaluronan chains become connected by hydrogen bonds when the pH is changed from 7.0 to 2.5 and that the bond density at pH 2.5 is independent of temperature. Temperature-dependent rheology measurements show that because of this hydrogen bonding the stress relaxation at pH 2.5 is strongly slowed down in comparison to pH 7.0, consistent with the sticky reptation model of associative polymers. From the flow activation energy, we conclude that each polymer is cross-linked by multiple (5-15) hydrogen bonds to others, causing slow macroscopic stress relaxation, despite the short time scale of breaking and reformation of each individual hydrogen bond. Our findings can aid the design of stimuli-responsive hydrogels with tailored viscoelastic properties for biomedical applications.

Hyaluronic acid is an abundant polyelectrolyte in the human body that forms extracellular hydrogels in connective tissues. It is essential for regulating tissue biomechanics and cell-cell communication, yet hyaluronan overexpression is associated with pathological situations such as cancer and multiple sclerosis. Due to its enormous molecular weight (in the range of millions of Daltons), accumulation of hyaluronan hinders transport of macromolecules including nutrients and growth factors through tissues and also hampers drug delivery. However, the exact contribution of hyaluronan to tissue penetrability is poorly understood due to the complex structure and molecular composition of tissues. Here we reconstitute biomimetic hyaluronan gels and systematically investigate the effects of gel composition and crosslinking on the diffusion of microscopic tracer particles. We combine ensemble-averaged measurements via differential dynamic microscopy with single-particle tracking. We show that the particle diffusivity depends on the particle size relative to the network pore size and also on the stress relaxation dynamics of the network. We furthermore show that addition of collagen, the other major biopolymer in tissues, causes the emergence of caged particle dynamics. Our findings are useful for understanding macromolecular transport in tissues and for designing biomimetic extracellular matrix hydrogels for drug delivery and tissue regeneration.

Bottom-up biology is an expanding research field that aims to understand the mechanisms underlying biological processes via in vitro assembly of their essential components in synthetic cells. As encapsulation and controlled manipulation of these elements is a crucial step in the recreation of such cell-like objects, microfluidics is increasingly used for the production of minimal artificial containers such as single-emulsion droplets, double-emulsion droplets, and liposomes. Despite the importance of cell morphology on cellular dynamics, current synthetic-cell studies mainly use spherical containers, and methods to actively shape manipulate these have been lacking. In this paper, we describe a microfluidic platform to deform the shape of artificial cells into a variety of shapes (rods and discs) with adjustable cell-like dimensions below 5 μm, thereby mimicking realistic cell morphologies. To illustrate the potential of our method, we reconstitute three biologically relevant protein systems (FtsZ, microtubules, collagen) inside rod-shaped containers and study the arrangement of the protein networks inside these synthetic containers with physiologically relevant morphologies resembling those found in living cells.