Connecting the Stimuli-Responsive Rheology of Biopolymer Hydrogels to Underlying Hydrogen-Bonding Interactions.

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.

Hyaluronan biopolymers release water upon pH-induced gelation.

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.

Connectivity and plasticity determine collagen network fracture.

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.

Particle diffusion in extracellular hydrogels.

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.

Revealing the molecular origins of fibrin's elastomeric properties by in situ X-ray scattering.

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.

Revealing the assembly of filamentous proteins with scanning transmission electron microscopy.

Filamentous proteins are responsible for the superior mechanical strength of our cells and tissues. The remarkable mechanical properties of protein filaments are tied to their complex molecular packing structure. However, since these filaments have widths of several to tens of nanometers, it has remained challenging to quantitatively probe their molecular mass density and three-dimensional packing order. Scanning transmission electron microscopy (STEM) is a powerful tool to perform simultaneous mass and morphology measurements on filamentous proteins at high resolution, but its applicability has been greatly limited by the lack of automated image processing methods. Here, we demonstrate a semi-automated tracking algorithm that is capable of analyzing the molecular packing density of intra- and extracellular protein filaments over a broad mass range from STEM images. We prove the wide applicability of the technique by analyzing the mass densities of two cytoskeletal proteins (actin and microtubules) and of the main protein in the extracellular matrix, collagen. The high-throughput and spatial resolution of our approach allow us to quantify the internal packing of these filaments and their polymorphism by correlating mass and morphology information. Moreover, we are able to identify periodic mass variations in collagen fibrils that reveal details of their axially ordered longitudinal self-assembly. STEM-based mass mapping coupled with our tracking algorithm is therefore a powerful technique in the characterization of a wide range of biological and synthetic filaments.

Supercoiling DNA optically.

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

Shape and Size Control of Artificial Cells for Bottom-Up Biology.

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.

Molecular Origin of the Elastic State of Aqueous Hyaluronic Acid.

The macroscopic mechanical properties of biological hydrogels are broadly studied and successfully mimicked in synthetic materials, but little is known about the molecular interactions that mediate these properties. Here, we use two-dimensional infrared spectroscopy to study the pH-induced gelation of hyaluronic acid, a ubiquitous biopolymer, which undergoes a transition from a viscous to an elastic state in a narrow pH range around 2.5. We find that the gelation originates from the enhanced formation of strong interchain connections, consisting of a double amide-COOH hydrogen bond and an N-D-COO- hydrogen bond on the adjacent sugars of the hyaluronan disaccharide unit. We confirm the enhanced interchain connectivity in the elastic state by atomic force microscopy imaging.

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion.

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.