Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3ß3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by ß-ß interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the ß subunit interfaces, with subsequent targeted improvements of the subunits.

Superinsulating nanocellulose aerogels: Effect of density and nanofiber alignment.

Cellulose aerogels are potential alternatives to silica aerogels with advantages in cost, sustainability and mechanical properties. However, the density dependence of thermal conductivity (λ) for cellulose aerogels remains controversial. Cellulose aerogels were produced by gas-phase pH induced gelation of TEMPO-oxidized cellulose nanofibers (CNF) and supercritical drying. Their properties are evaluated by varying the CNF concentration (5-33 mg·cm-3) and by uniaxial compression (9-115 mg·cm-3). The aerogels are transparent with specific surface areas of ~400 m2·g-1, mesopore volumes of ~2 cm3·g-1 and a power-law dependence of the E-modulus (α ~ 1.53, and the highest reported E of ~1 MPa). The dataset confirms that λ displays a traditional U-shaped density dependence with a minimum of 18 mW·m-1·K-1 at 0.065 g·cm-3. For a given density, λ is ~5 mW·m-1·K-1 lower for compressed aerogels due to the alignment of nanofibers, confirmed by small angle X-ray scattering (SAXS).

Redesigned Hybrid Nylons with Optical Clarity and Chemical Recyclability.

Aliphatic polyamides, or nylons, are typically highly crystalline and thermally robust polymers used in high-performance applications. Nylon 6, a high-ceiling-temperature (HCT) polyamide from ε-caprolactam, lacks expedient chemical recyclability, while low-ceiling temperature (LCT) nylon 4 from pyrrolidone exhibits complete chemical recyclability, but it is thermally unstable and not melt-processable. Here, we introduce a hybrid nylon, nylon 4/6, based on a bicyclic lactam composed of both HCT ε-caprolactam and LCT pyrrolidone motifs in a hybridized offspring structure. Hybrid nylon 4/6 overcomes trade-offs in (de)polymerizability and performance properties of the parent nylons, exhibiting both excellent polymerization and facile depolymerization characteristics. This stereoregular polyamide forms nanocrystalline domains, allowing optical clarity and high thermal stability, however, without displaying a melting transition before decomposition. Of a series of statistical copolymers comprising nylon 4/6 and nylon 4, a 50/50 copolymer achieves the greatest synergy in both reactivity and polymer properties of each homopolymer, offering an amorphous nylon with favorable properties, including optical clarity, a high glass transition temperature, melt processability, and full chemical recyclability.

Understanding multiscale structure-property correlations in PVDF-HFP electrospun fiber membranes by SAXS and WAXS.

Electrospinning is a versatile technique to produce nanofibrous membranes with applications in filtration, biosensing, biomedical and tissue engineering. The structural and therefore physical properties of electrospun fibers can be finely tuned by changing the electrospinning parameters. The large parameter window makes it challenging to optimize the properties of fibers for a specific application. Therefore, a fundamental understanding of the multiscale structure of fibers and its correlation with their macroscopic behaviors is required for the design and production of systems with dedicated applications. In this study, we demonstrate that the properties of poly(vinylidene fluoride-co-hexafluoro propylene) (PVDF-HFP) electrospun fibers can be tuned by changing the rotating drum speed used as a collector during electrospinning. Indeed, with the help of multiscale characterization techniques such as scanning electron microscopy (SEM), small-angle X-ray scattering (SAXS), and wide-angle X-ray scattering (WAXS), we observe that increasing the rotating drum speed not only aligns the fibers but also induces polymeric chain rearrangements at the molecular scale. Such changes result in enhanced mechanical properties and an increase of the piezoelectric ß-phase of the PVDF-HFP fiber membranes. We detect nanostructural deformation behaviors when the aligned fibrous membrane is uniaxially stretched along the fiber alignment direction, while an increase in the alignment of the fibers is observed for randomly aligned samples. This was analyzed by performing in situ SAXS measurements coupled with uniaxial tensile loading of the fibrous membranes along the fiber alignment direction. The present study shows that fibrous membranes can be produced with varying degrees of fiber orientation, piezoelectric ß-phase content, and mechanical properties by controlling the speed of the rotating drum collector during the fiber production. Such aligned fiber membranes have potential applications for neural or musculoskeletal tissue engineering.

Tailoring Fibre Structure Enabled by X-ray Analytics for Targeted Biomedical Applications.

The rising interest in designing fibres via spinning techniques combining the properties of various polymeric materials into advanced functionalised materials is directed towards targeted biomedical applications such as drug delivery, wearable sensors or tissue engineering. Understanding how these functional polymers exhibit multiscale structures ranging from the molecular level to nano-, micro-and millimetre scale is a key prerequisite for their challenging applications that can be addressed by a non-destructive X-ray based analytical approach. X-ray multimodalities combining X-ray imaging, scattering and diffraction allow the study of morphology, molecular structure, and the analysis of nano-domain size and shape, crystallinity and preferential orientation in 3D arrangements. The incorporation of X-ray analytics in the design process of polymeric fibers via their nanostructure under non-ambient conditions (i.e. temperature, mechanical load, humidity…) allows for efficient optimization of the fabrication process as well as quality control along the product lifetime under operating environmental conditions. Here, we demonstrate the successful collaboration between the laboratory of Biomimetic Textiles and Membranes and the Center of X-ray Analytics at Empa for the design, characterisation and optimisation of advanced functionalised polymeric fibrous material systems.

Unraveling the Influence of Thermal Drawing Parameters on the Microstructure and Thermo-Mechanical Properties of Multimaterial Fibers.

Multimaterial thermally drawn fibers are becoming important building blocks in several foreseen applications in surgical probes, protective gears, or medical textiles. Here, the influence of the thermal drawing parameters on the degree of polymer chain orientation, the related thermal shrinkage behavior, and the mechanical properties of the final fibers is investigated via thermo-mechanical testing and small- and wide-angle X-ray scattering (SAXS and WAXS) analyses. This study on polyetherimide fibers reveals that the drawing stress, which depends on the drawing speed and temperature, controls the thermal shrinkage behavior and mechanical properties. Furthermore, SAXS and WAXS analyses show that the degree of chain orientation increases with drawing stresses below 8 MPa and then saturates, which correlates with the amount of observed shrinkage. The use of this process-dependent polymer chain alignment to tune the mechanical and shrinkage properties of the fibers is highlighted and controlled bending multimaterial fibers made of two polymethyl methacrylates having different molecular weights are developed. Finally, a heat treatment procedure is proposed to relax the chain alignment and increase the dimensional stability of devices such as temperature sensors. This deeper understanding can serve as a guide for the processing of complex fibers requiring specific mechanical properties or enhanced thermal stability.

Multiscale and multimodal X-ray analysis: Quantifying phase orientation and morphology of mineralized turkey leg tendons.

Fibrous biocomposites like bone and tendons exhibit a hierarchical arrangement of their components ranging from the macroscale down to the molecular level. The multiscale complex morphology, together with the correlated orientation of their constituents, contributes significantly to the outstanding mechanical properties of these biomaterials. In this study, a systematic road map is provided to quantify the hierarchical structure of a mineralized turkey leg tendon (MTLT) in a holistic multiscale evaluation by combining micro-Computed Tomography (micro-CT), small-angle X-ray scattering (SAXS), and wide-angle X-ray diffraction (WAXD). We quantify the interplay of the main MTLT components with respect to highly ordered organic parts such as fibrous collagen integrating inorganic components like hydroxyapatite (HA). The microscale fibrous morphology revealing different types of porous features and their orientation was quantified based on micro-CT investigations. The quantitative analysis of the alignment of collagen fibrils and HA crystallites was established from the streak-like signal in SAXS using the Ruland approach and the broadening of azimuthal profiles of the small and wide-angle diffraction peaks. It has been in general agreement that HA crystallites are co-aligned with the nanostructure of mineralized tissue. However, we observe relatively lower degree of orientation of HA crystallites compared to the collagen fibrils, which supports the recent findings of the structural interrelations within mineralized tissues. The generic multiscale characterization approach of this study is relevant to any hierarchically structured biomaterials or bioinspired materials from the µm-nm-Å scale. Hence, it gives the basis for future structure-property relationship investigations and simulations for a wide range of hierarchically structured materials. STATEMENT OF SIGNIFICANCE: Many fibrous biocomposites such as tendon, bone, and wood possess multiscale hierarchical structures, responsible for their exceptional mechanical properties. In this study, the 3-dimensional hierarchical structure, the degree of orientation and composition of mineralized tendon extracted from a turkey leg were quantified using a multimodal X-ray based approach combining small-angle X-ray scattering and wide-angle X-ray diffraction with micro-Computed Tomography. We demonstrate that hydroxyapatite (HA) domains are co-aligned with the nanostructure of mineralized tissue. However, the lower degree of orientation of HA crystallites was observed when compared to the collagen fibrils. The generic multiscale characterization approach of this study is relevant to any hierarchically structured biomaterials or bioinspired materials from the micrometer over the nanometer to the Angström scale level.

Combining polarized Raman spectroscopy and micropillar compression to study microscale structure-property relationships in mineralized tissues.

Bone is a natural composite possessing outstanding mechanical properties combined with a lightweight design. The key feature contributing to this unusual combination of properties is the bone hierarchical organization ranging from the nano- to the macro-scale. Bone anisotropic mechanical properties from two orthogonal planes (along and perpendicular to the main bone axis) have already been widely studied. In this work, we demonstrate the dependence of the microscale compressive mechanical properties on the angle between loading direction and the mineralized collagen fibril orientation in the range between 0° and 82°. For this, we calibrated polarized Raman spectroscopy for quantitative collagen fibril orientation determination and validated the method using widely used techniques (small angle X-ray scattering, micro-computed tomography). We then performed compression tests on bovine cortical bone micropillars with known mineralized collagen fibril angles. A strong dependence of the compressive micromechanical properties of bone on the fibril orientation was found with a high degree of anisotropy for both the elastic modulus (Ea/Et=3.80) and the yield stress (σay/σty=2.54). Moreover, the post-yield behavior was found to depend on the MCF orientation with a transition between softening to hardening behavior at approximately 50°. The combination of methods described in this work allows to reliably determine structure-property relationships of bone at the microscale, which may be used as a measure of bone quality.

Responsive Nanofibers with Embedded Hierarchical Lipid Self-Assemblies.

We introduce the design and study of a hybrid electrospun membrane with a dedicated nanoscale structural hierarchy for controlled functions in the biomedical domain. The hybrid system comprises submicrometer-sized internally self-assembled lipid nanoparticles (ISAsomes or mesosomes) embedded into the electrospun membrane with a nanofibrous polymer network. The internal structure of ISAsomes, studied by small-angle X-ray scattering (SAXS) and electron microscopy, demonstrated a spontaneous response to variations in the environmental conditions as they undergo a bicontinuous inverse cubic phase (cubosomes) in solution to a crystalline lamellar phase in the polymer membrane; nevertheless, this phase reorganization is reversible. As revealed by in situ SAXS measurements, if the membrane was put in contact with aqueous media, the cubic phase reappeared and submicrometer-sized cubosomes were released upon dissolution of the nanofibers. Furthermore, the hybrid membranes exhibited a specific anisotropic feature and morphological response under an external strain. While nanofibers were aligned under external strain in the microscale, the semicrystalline domains from the polymer phase were positioned perpendicular to the lamellae of the lipid phase in the nanoscale. The fabricated membranes and their spontaneous responses offer new strategies for the development of structure-controlled functions in electrospun nanofibers for biomedical applications, such as drug delivery or controlled interactions with biointerfaces.

Polarimetric imaging in backscattering for the structural characterization of strongly scattering birefringent fibrous media.

Interpreting the polarimetric data from fiber-like macromolecules constitutive of tissue can be difficult due to strong scattering. In this study, we probed the superficial layers of fibrous tissue models (membranes consisting of nanofibers) displaying varying degrees of alignment. To better understand the manifestation of membranes' degree of alignment in polarimetry, we analyzed the spatial variations of the backscattered light's Stokes vectors as a function of the orientation of the probing beam's linear polarization. The degree of linear polarization reflects the uniaxially birefringent behavior of the membranes. The rotational (a-)symmetry of the backscattered light's degree of linear polarization provides a measure of the membranes' degree of alignment.

Structural insights into semicrystalline states of electrospun nanofibers: a multiscale analytical approach.

A dedicated nanofiber design for applications in the biomedical domain is based on the understanding of nanofiber structures. The structure of electrospun nanofibers strongly influences their properties and functionalities. In polymeric nanofibers X-ray scattering and diffraction methods, i.e. SAXS and WAXD, are capable of decoding their structural insights from about 100 nm down to the Angström scale. Here, we present a comprehensive X-ray scattering and diffraction based study and introduce new data analysis approaches to unveil detailed structural features in electrospun poly(vinylidene fluoride-co-hexafluoropropylene) (PVDFhfp) nanofiber membranes. Particular emphasis was placed on anisotropic morphologies being developed during the nanofiber fabrication process. Global analysis was performed on SAXS data to derive the nanofibrillar structure of repeating lamella crystalline domains with average dimensions of 12.5 nm thickness and 7.8 nm spacing along with associated tie-molecules. The varying surface roughness of the nanofiber was evaluated by extracting the Porod exponent in parallel and perpendicular direction to the nanofiber axis, which was further validated by Atomic Force Microscopy. Additionally, the presence of a mixture of the monoclinic alpha and the orthorhombic beta PVDFhfp phases both exhibiting about 6% larger unit cells compared to the corresponding pure PVDF phases was derived from WAXD. The current study shows a generic approach in detailed understanding of internal structures and surface morphology for nanofibers. This forms the basis for targeted structure and morphology steering and the respective controlling during the fabrication process with the aim to engineer nanofibers for different biomedical applications with specific requirements.