Structure-Property Relationships of Elastin-like Polypeptides: A Review of Experimental and Computational Studies.

Elastin is a structural protein with outstanding mechanical properties (e.g., elasticity and resilience) and biologically relevant functions (e.g., triggering responses like cell adhesion or chemotaxis). It is formed from its precursor tropoelastin, a 60-72 kDa water-soluble and temperature-responsive protein that coacervates at physiological temperature, undergoing a phenomenon termed lower critical solution temperature (LCST). Inspired by this behavior, many scientists and engineers are developing recombinantly produced elastin-inspired biopolymers, usually termed elastin-like polypeptides (ELPs). These ELPs are generally comprised of repetitive motifs with the sequence VPGXG, which corresponds to repeats of a small part of the tropoelastin sequence, X being any amino acid except proline. ELPs display LCST and mechanical properties similar to tropoelastin, which renders them promising candidates for the development of elastic and stimuli-responsive protein-based materials. Unveiling the structure-property relationships of ELPs can aid in the development of these materials by establishing the connections between the ELP amino acid sequence and the macroscopic properties of the materials. Here we present a review of the structure-property relationships of ELPs and ELP-based materials, with a focus on LCST and mechanical properties and how experimental and computational studies have aided in their understanding.

Sequence Control of the Self-Assembly of Elastin-Like Polypeptides into Hydrogels with Bespoke Viscoelastic and Structural Properties.

The biofabrication of structural proteins with controllable properties via amino acid sequence design is interesting for biomedicine and biotechnology, yet a complete framework that connects amino acid sequence to material properties is unavailable, despite great progress to establish design rules for synthesizing peptides and proteins with specific conformations (e.g., unfolded, helical, ß-sheets, or ß-turns) and intermolecular interactions (e.g., amphipathic peptides or hydrophobic domains). Molecular dynamics (MD) simulations can help in developing such a framework, but the lack of a standardized way of interpreting the outcome of these simulations hinders their predictive value for the design of de novo structural proteins. To address this, we developed a model that unambiguously classifies a library of de novo elastin-like polypeptides (ELPs) with varying numbers and locations of hydrophobic/hydrophilic and physical/chemical-cross-linking blocks according to their thermoresponsiveness at physiological temperature. Our approach does not require long simulation times or advanced sampling methods. Instead, we apply (un)supervised data analysis methods to a data set of molecular properties from relatively short MD simulations (150 ns). We also experimentally investigate hydrogels of those ELPs from the library predicted to be thermoresponsive, revealing several handles to tune their mechanical and structural properties: chain hydrophilicity/hydrophobicity or block distribution control the viscoelasticity and thermoresponsiveness, whereas ELP concentration defines the network permeability. Our findings provide an avenue to accelerate the design of de novo ELPs with bespoke phase behavior and material properties.

Molecular simulations of the interfacial properties in silk-hydroxyapatite composites.

Biomineralization is a common strategy used in Nature to improve the mechanical strength and toughness of biological materials. This strategy, applied in materials like bone or nacre, serves as inspiration for materials scientists and engineers to design new materials for applications in healthcare, soft robotics or the environment. In this regard, composites consisting of silk and hydroxyapatite have been extensively researched for bone regeneration applications, due to their reported cytocompatibility and osteoinduction capacity that supports bone formation in vivo. Thus, it becomes relevant to understand how silk and hydroxyapatite interact at their interface, and how this affects the overall mechanical properties of these composites. This theoretical-experimental work investigates the interfacial dynamic and structural properties of silk in contact with hydroxyapatite, combining molecular dynamics simulations with analytical characterization. Our data indicate that hydroxyapatite decreases the ß-sheets in silk, which are a key load-bearing element of silk. The ß-sheets content can usually be increased in silk biomaterials via post-processing methods, such as water vapor annealing. However, the presence of hydroxyapatite appears to reduce also for the formation of ß-sheets via water vapor annealing. This work sheds light into the interfacial properties of silk-hydroxyapatite composite and their relevance for the design of composite biomaterials for bone regeneration.

Effect of the silica nanoparticle size on the osteoinduction of biomineralized silk-silica nanocomposites.

Understanding the properties and behavior of biomineralized protein-based materials at the organic-inorganic interface is critical to optimize the performance of such materials for biomedical applications. To that end, this work investigates biomineralized protein-based films with applications for bone regeneration. These films were generated using a chimeric protein fusing the consensus repeat derived from the spider Nephila clavipes major ampullate dragline silk with the silica-promoting peptide R5 derived from the Cylindrotheca fusiformis silaffin gene. The effect of pH on the size of silica nanoparticles during their biomineralization on silk films was investigated, as well as the potential impact of nanoparticle size on the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts. To that end, induction of the integrin αV subunit and the osteogenic markers Runx2 transcription factor and Bone Sialoprotein (BSP) was followed. The results indicated that pH values of 7-8 during biomineralization maximized the coverage of the film surface by silica nanoparticles yielding nanoparticles ranging 200-500 nm and showing enhanced osteoinduction in gene expression analysis. Lower (3-5) or high (10) pH values led to lower biomineralization and poor coverage of the protein surfaces, showing reduced osteoinduction. Molecular dynamics simulations confirmed the activation of the integrin αVß3 in contact with silica nanoparticles, correlating with the experimental data on the induction of osteogenic markers. This work sheds light on the optimal conditions for the development of fit-for-purpose biomaterial designs for bone regeneration, while the agreement between experimental and computational results shows the potential of computational methods to predict the expression of osteogenic markers for biomaterials. STATEMENT OF SIGNIFICANCE: The ability of biomineralized materials to induce hMSCs differentiation for bone tissue regeneration applications was analyzed. Biomaterials were created using a recombinant protein formed by the consensus repeat derived from the spider Nephila clavipes major ampullate dragline silk and the silica-promoting peptide R5 derived from the Cylindrotheca fusiformis silaffin gene. A combination of computational and experimental techniques revealed the optimal conditions for the synthesis of biomineralized silk-silica films with enhanced expression of markers related to bone regeneration.

Conductive Silk-Based Composites Using Biobased Carbon Materials.

There is great interest in developing conductive biomaterials for the manufacturing of sensors or flexible electronics with applications in healthcare, tracking human motion, or in situ strain measurements. These biomaterials aim to overcome the mismatch in mechanical properties at the interface between typical rigid semiconductor sensors and soft, often uneven biological surfaces or tissues for in vivo and ex vivo applications. Here, the use of biobased carbons to fabricate conductive, highly stretchable, flexible, and biocompatible silk-based composite biomaterials is demonstrated. Biobased carbons are synthesized via hydrothermal processing, an aqueous thermochemical method that converts biomass into a carbonaceous material that can be applied upon activation as conductive filler in composite biomaterials. Experimental synthesis and full-atomistic molecular dynamics modeling are combined to synthesize and characterize these conductive composite biomaterials, made entirely from renewable sources and with promising applications in fields like biomedicine, energy, and electronics.

Multiscale Modeling of Silk and Silk-Based Biomaterials-A Review.

Silk embodies outstanding material properties and biologically relevant functions achieved through a delicate hierarchical structure. It can be used to create high-performance, multifunctional, and biocompatible materials through mild processes and careful rational material designs. To achieve this goal, computational modeling has proven to be a powerful platform to unravel the causes of the excellent mechanical properties of silk, to predict the properties of the biomaterials derived thereof, and to assist in devising new manufacturing strategies. Fine-scale modeling has been done mainly through all-atom and coarse-grained molecular dynamics simulations, which offer a bottom-up description of silk. In this work, a selection of relevant contributions of computational modeling is reviewed to understand the properties of natural silk, and to the design of silk-based materials, especially combined with experimental methods. Future research directions are also pointed out, including approaches such as 3D printing and machine learning, that may enable a high throughput design and manufacturing of silk-based biomaterials.

The Rise of Hierarchical Nanostructured Materials from Renewable Sources: Learning from Nature.

Mimicking Nature implies the use of bio-inspired hierarchical designs to manufacture nanostructured materials. Such materials should be produced from sustainable sources ( e.g., biomass) and through simple processes that use mild conditions, enabling sustainable solutions. The combination of different types of nanomaterials and the implementation of different features at different length scales can provide synthetic hierarchical nanostructures that mimic natural materials, outperforming the properties of their constitutive building blocks. Taking recent developments in flow-assisted assembly of nanocellulose crystals as a starting point, we review the state of the art and provide future perspectives on the manufacture of hierarchical nanostructured materials from sustainable sources, assembly techniques, and potential applications.

Sub- and supercritical water oxidation of anaerobic fermentation sludge for carbon and nitrogen recovery in a regenerative life support system.

Sub- and supercritical water oxidation was applied to recover carbon as CO2, while maintaining nitrogen as NH4+ or NO3-, from sludge obtained from an anaerobic fermenter running on a model waste composed of plant residues and human fecal matter. The objective was to fully convert carbon in the organic waste to CO2 while maintaining nutrients (specifically N) in the liquid effluent. In regenerative life support systems, CO2 and nutrients could then be further used in plant production; thus creating a closed carbon and nutrient cycle. The effect of the operational parameters in water oxidation on carbon recovery (C-to-CO2) and nitrogen conversion (to NH4+, NO3-) was investigated. A batch micro-autoclave reactor was used, at pressures ranging between 110 and 300 bar and at temperatures of 300-500 °C using hydrogen peroxide as oxidizer. Residence times of 1, 5 and 10 min were tested. Oxidation efficiency increased as temperature increased, with marginal improvements beyond the critical temperature of water. Prolonging the residence time improved only slightly the carbon oxidation efficiency. Adequate oxygen supply, i.e., exceeding the stoichiometrically required amount, resulted in high carbon conversion efficiencies (>85%) and an odorless, clear liquid effluent. However, the corresponding oxidizer use efficiency was low, up to 50.2% of the supplied oxygen was recovered as O2 in the effluent gas and did not take part in the oxidation. Volatile fatty acids (VFAs) were found as the major soluble organic compounds remaining in the effluent liquid. Nitrogen recovery was high at 1 min residence time (>94.5%) and decreased for longer residence times (down to 36.4% at 10 min). Nitrogen in the liquid effluent was mostly in the form of ammonium.

Assessing microalgae biorefinery routes for the production of biofuels via hydrothermal liquefaction.

The interest in third generation biofuels from microalgae has been rising during the past years. Meanwhile, it seems not economically feasible to grow algae just for biofuels. Co-products with a higher value should be produced by extracting a particular algae fraction to improve the economics of an algae biorefinery. The present study aims at analyzing the influence of two main microalgae components (lipids and proteins) on the composition and quantity of biocrude oil obtained via hydrothermal liquefaction of two strains (Nannochloropsis gaditana and Scenedesmus almeriensis). The algae were liquefied as raw biomass, after extracting lipids and after extracting proteins in microautoclave experiments at different temperatures (300-375°C) for 5 and 15min. The results indicate that extracting the proteins from the microalgae prior to HTL may be interesting to improve the economics of the process while at the same time reducing the nitrogen content of the biocrude oil.

Influence of strain-specific parameters on hydrothermal liquefaction of microalgae.

Algae are an interesting feedstock for producing biofuel via hydrothermal liquefaction (HTL), due to their high water content. In this study, algae slurries (5-7 wt% daf) from different species were liquefied at 250 and 375 °C in batch autoclaves during 5 min. The aim was to analyze the influence of strain-specific parameters (cell structure, biochemical composition and growth environment) on the HTL process. Results show big variations in the biocrude oil yield within species at 250 °C (from 17.6 to 44.8 wt%). At 375 °C, these differences become less significant (from 45.6 to 58.1 wt%). An appropriate characterization of feedstock appeared to be critical to interpret the results. If a high conversion of microalgae-to-biocrude is pursued, near critical conditions are required, with Scenedesmus almeriensis (freshwater) and Nannochloropsis gaditana (marine) leading to the biocrude oils with lower nitrogen content from each growth environment.