Sustainable Bombyx mori's silk fibroin for biomedical applications as a molecular biotechnology challenge: A review.

Silk is a natural engineering material with a unique set of properties. The major constituent of silk is fibroin, a protein widely used in the biomedical field because of its mechanical strength, toughness and elasticity, as well as its biocompatibility and biodegradability. The domestication of silkworms allows large amounts of fibroin to be extracted inexpensively from silk cocoons. However, the industrial extraction process has drawbacks in terms of sustainability and the quality of the final medical product. The heterologous production of fibroin using recombinant DNA technology is a promising approach to address these issues, but the production of such recombinant proteins is challenging and further optimization is required due to the large size and repetitive structure of fibroin's DNA and amino acid sequence. In this review, we describe the structure-function relationship of fibroin, the current extraction process, and some insights into the sustainability of silk production for biomedical applications. We focus on recent advances in molecular biotechnology underpinning the production of recombinant fibroin, working toward a standardized, successful and sustainable process.

Manufacturing of scaffolds with interconnected internal open porosity and surface roughness.

Manufacturing of three-dimensional scaffolds with multiple levels of porosity are an advantage in tissue regeneration approaches to influence cell behavior. Three-dimensional scaffolds with surface roughness and intra-filament open porosity were successfully fabricated by additive manufacturing combined with chemical foaming and porogen leaching without the need of toxic solvents. The decomposition of sodium citrate, a chemical blowing agent, generated pores within the scaffold filaments, which were interconnected and opened to the external environment by leaching of a water-soluble sacrificial phase, as confirmed by micro-CT and buoyancy measurements. The additional porosity did not result in lower elastic modulus, but in higher strain at maximum load, i.e. scaffold ductility. Human mesenchymal stromal cells cultured for 24 h adhered in greater numbers on these scaffolds when compared to plain additive-manufactured ones, irrespectively of the scaffold pre-treatment method. Additionally, they showed a more spread and random morphology, which is known to influence cell fate. Cells cultured for a longer period exhibited enhanced metabolic activity while secreting higher osteogenic markers after 7 days in culture. STATEMENT OF SIGNIFICANCE: Inspired by the function of hierarchical cellular structures in natural materials, this work elucidates the development of scaffolds with multiscale porosity by combining in-situ foaming and additive manufacturing, and successive porogen leaching. The resulting scaffolds displayed enhanced mechanical toughness and multiscale pore network interconnectivity, combined with early differentiation of adult mesenchymal stromal cells into the osteogenic lineage.

Hydration, Refinement, and Dissolution of the Crystalline Phase in Polyamide 6 Polymorphs for Ultimate Thermomechanical Properties.

Timescales of polyamide 6 melt-shaping technologies, relative to the dynamics of conformational rearrangements upon crystallization, challenge the formation of the most thermodynamically favorable chain packing and thus optimum performance. In this publication, we make use of the mediation of hydrogen bonding by water molecules in the superheated state of water, i.e., above 100 °C in a closed environment, in the structural refinement of polyamide 6 for enhanced thermomechanical performance. The paper addresses dissolution and (re)crystallization of different polyamide 6 polymorphs in the superheated state of water by time-resolved simultaneous small- and wide-angle X-ray scattering and solid-state 1H NMR spectroscopy and the effect on mechanical properties. The experiments reveal that upon heating in the superheated state of water, the pseudo-hexagonal phase dissolves at relatively low temperature and instantly crystallizes in a defected monoclinic phase that successively refines to a perfected monoclinic structure. The dissolution temperature of the pseudo-hexagonal phase of polyamide 6 is found to be dependent on the degree of crystal perfection originating from conformational disorder and misalignment of hydrogen bonding in the lattice, retrospectively, to the Brill transition temperature. The perfected monoclinic phase below the dissolution temperature can be preserved upon cooling but is plasticized by hydration of the amide moieties in the crystalline phase. The removal of water from the hydrated crystals, in the proximity of Brill transition temperature, strengthening the hydrogen bonding, occurs. Retrospectively, the most thermodynamically stable crystallographic phase is preserved and renders an increase in mechanical properties and dimensional stability of the product. The insight obtained on the influence of superheated water on the structural refinement of imperfected crystallographic states assists in polyamide 6 postprocessing strategies for enhanced performance.

Cholecalciferol as Bioactive Plasticizer of High Molecular Weight Poly(D,L-Lactic Acid) Scaffolds for Bone Regeneration.

Synthetic thermoplastic polymers are a widespread choice as material candidates for scaffolds for tissue engineering (TE), thanks to their ease of processing and tunable properties with respect to biological polymers. These features made them largely employed in melt-extrusion-based additive manufacturing, with particular application in hard-TE. In this field, high molecular weight (Mw) polymers ensuring entanglement network strength are often favorable candidates as scaffold materials because of their enhanced mechanical properties compared with lower Mw grades. However, this is accompanied by high viscosities once processed in molten conditions, which requires driving forces not always accessible technically or compatible with often chemically nonstabilized biomedical grades. When possible, this is circumvented by increasing the operating temperature, which often results in polymer chain scission and consequent degradation of properties. In addition, synthetic polymers are mostly considered bioinert compared with biological materials, and additional processing steps are often required to make them favorable for tissue regeneration. In this study, we report the plasticization of a common thermoplastic polymer with cholecalciferol, the metabolically inactive form of vitamin D3 (VD3). Plasticization of the polymer allowed us to reduce its melt viscosity, and therefore the energy requirements (mechanical [torque] and heat [temperature]) for extrusion, limiting ultimately polymer degradation. In addition, we evaluated the effect of cholecalciferol, which is more easily available than its active counterpart, on the osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Results indicated that cholecalciferol supported osteogenic differentiation more than the osteogenic culture medium, suggesting that hMSCs possess the enzymatic toolbox for VD3 metabolism. Impact statement Limitations in mechanical and biological performances of scaffolds manufactured through melt deposition may result from material thermal degradation during processing and inherent bioinertness of synthetic polymers. Current approaches involve the incorporation of chemical additives to reduce the extent of thermal degradation, which are often nonbiocompatible or may lead to uncontrolled modifications to the polymer structure. Lack of polymer bioactivity is tackled by postfunctionalization methods that often involve extra processes extending scaffold production time. Therefore, new methods to improve scaffolds performances should consider preserving the integrity of the molecular structure and improving biological responsiveness of the material while keeping the process as straightforward as possible.

Additive Manufacturing of α-Amino Acid Based Poly(ester amide)s for Biomedical Applications.

α-Amino acid based polyester amides (PEAs) are promising candidates for additive manufacturing (AM), as they unite the flexibility and degradability of polyesters and good thermomechanical properties of polyamides in one structure. Introducing α-amino acids in the PEA structure brings additional advantages such as (i) good cytocompatibility and biodegradability, (ii) providing strong amide bonds, enhancing the hydrogen-bonding network, (iii) the introduction of pendant reactive functional groups, and (iv) providing good cell-polymer interactions. However, the application of α-amino acid based PEAs for AM via fused deposition modeling (FDM), an important manufacturing technique with unique processing characteristics and requirements, is still lacking. With the aim to exploit the combination of these advantages in the creation, design, and function of additively manufactured scaffolds using FDM, we report the structure-function relationship of a series of α-amino acid based PEAs. The PEAs with three different molecular weights were synthesized via the active solution polycondensation, and their performance for AM applications was studied in comparison with a commercial biomedical grade copolymer of l-lactide and glycolide (PLGA). The PEAs, in addition to good thermal stability, showed semicrystalline behavior with proper mechanical properties, which were different depending on their molecular weight and crystallinity. They showed more ductility due to their lower glass transition temperature (Tg; 18-20 °C) compared with PLGA (57 °C). The rheology studies revealed that the end-capping of PEAs is of high importance for preventing cross-linking and further polymerization during the melt extrusion and for the steadiness and reproducibility of FDM. Furthermore, our data regarding the steady 3D printing performance, good polymer-cell interactions, and low cytotoxicity suggest that α-amino acid based PEAs can be introduced as favorable polymers for future AM applications in tissue engineering. In addition, their ability for formation of bonelike apatite in the simulated body fluid (SBF) indicates their potential for bone tissue engineering applications.

Topographical Vacuum Sealing of 3D-Printed Multiplanar Microfluidic Structures.

We demonstrate a novel way of creating three-dimensional microfluidic channels capable of following complex topographies. To this end, substrates with open channels and different geometries were 3D-printed, and the open channels were consecutively closed with a thermoplastic using a low-resolution vacuum-forming approach. This process allows the sealing of channels that are located on the surface of complex multiplanar topographies, as the thermoplastic aligns with the surface-shape (the macrostructure) of the substrate, while the microchannels remain mostly free of thermoplastic as their small channel size resists thermoplastic inflow. This new process was analyzed for its capability to consistently close different substrate geometries, which showed reliable sealing of angles >90°. Furthermore, the thermoplastic intrusion into channels of different widths was quantified, showing a linear effect of channel width and percentage of thermoplastic intrusion; ranging from 43.76% for large channels with 2 mm width to only 5.33% for channels with 500 µm channel width. The challenging sealing of substrate 'valleys', which are created when two large protrusions are adjacent to each other, was investigated and the correlation between protrusion distance and height is shown. Lastly, we present three application examples: a serpentine mixer with channels spun around a cuboid, increasing the usable surface area; a cuvette-inspired flow cell for a 2-MXP biosensor based on molecular imprinted polymers, fitting inside a standard UV/Vis-Spectrophotometer; and an adapter system that can be manufactured by one-sided injection molding and is self-sealed before usage. These examples demonstrate how this novel technology can be used to easily adapt microfluidic circuits for application in biosensor platforms.

Additive Manufactured Scaffolds for Bone Tissue Engineering: Physical Characterization of Thermoplastic Composites with Functional Fillers.

Thermoplastic polymer-filler composites are excellent materials for bone tissue engineering (TE) scaffolds, combining the functionality of fillers with suitable load-bearing ability, biodegradability, and additive manufacturing (AM) compatibility of the polymer. Two key determinants of their utility are their rheological behavior in the molten state, determining AM processability and their mechanical load-bearing properties. We report here the characterization of both these physical properties for four bone TE relevant composite formulations with poly(ethylene oxide terephthalate)/poly(butylene terephthalate (PEOT/PBT) as a base polymer, which is often used to fabricate TE scaffolds. The fillers used were reduced graphene oxide (rGO), hydroxyapatite (HA), gentamicin intercalated in zirconium phosphate (ZrP-GTM) and ciprofloxacin intercalated in MgAl layered double hydroxide (MgAl-CFX). The rheological assessment showed that generally the viscous behavior dominated the elastic behavior (G″ > G') for the studied composites, at empirically determined extrusion temperatures. Coupled rheological-thermal characterization of ZrP-GTM and HA composites showed that the fillers increased the solidification temperatures of the polymer melts during cooling. Both these findings have implications for the required extrusion temperatures and bonding between layers. Mechanical tests showed that the fillers generally not only made the polymer stiffer but more brittle in proportion to the filler fractions. Furthermore, the elastic moduli of scaffolds did not directly correlate with the corresponding bulk material properties, implying composite-specific AM processing effects on the mechanical properties. Finally, we show computational models to predict multimaterial scaffold elastic moduli using measured single material scaffold and bulk moduli. The reported characterizations are essential for assessing the AM processability and ultimately the suitability of the manufactured scaffolds for the envisioned bone regeneration application.

Additive Manufacturing Using Melt Extruded Thermoplastics for Tissue Engineering.

Melt extrusion of thermoplastic materials is an important technique for fabricating tissue engineering scaffolds by additive manufacturing methods. Scaffold manufacturing is commonly achieved by one of the following extrusion-based techniques: fused deposition modelling (FDM), 3D-fiber deposition (3DF), and bioextrusion. FDM needs the input material to be strictly in the form of a filament, whereas 3DF and bioextrusion can be used to process input material in several forms, such as pellets or powder. This chapter outlines a common workflow for all these methods, going from the material to a scaffold, while highlighting the special requirements of particular methods. A few ways of characterizing the scaffolds are also briefly described.

Formation of cyclic structures in the cationic ring-opening polymerization of 1,3-dioxolane.

The cationic ring-opening polymerization of acetals is prone to cyclization of the polymer chains. This is also the case for the polymerization of 1,3-dioxolane. Literature states that this cyclization can be reduced by applying the Active Monomer mechanism, at least if no competition with the Active Chain End mechanism occurs. In this work, a detailed characterization of the different distributions resulting from the cationic ring-opening polymerization of 1,3-dioxolane via the Active Monomer mechanism is made by a combination of gel permeation chromatography, 1H NMR, and for the first time by matrix assisted laser desorption/ionization time of flight mass spectrometry. The influence of monomer addition speed, catalyst to initiator ratio and solvent were studied on both kinetics and composition of the product. Furthermore, it was found that increasing the conversion and monomer to initiator ratios leads to an increased amount of cyclic structures and to broader distributions, in correspondence with the Jacobson-Stockmayer theory. Furthermore, ion trapping experiments using 31P NMR provide insights into the actual reaction mechanism. Finally, purification of the products after the reactions led to a reduction of the cyclic fraction.

Additive manufacturing of an elastic poly(ester)urethane for cartilage tissue engineering.

Although a growing knowledge on the field of tissue engineering of articular cartilage exists, reconstruction or in-vitro growth of functional hyaline tissue still represents an unmet challenge. Despite the simplicity of the tissue in terms of cell population and absence of innervation and vascularization, the outstanding mechanical properties of articular cartilage, which are the result of the specificity of its extra cellular matrix (ECM), are difficult to mimic. Most importantly, controlling the differentiation state or phenotype of chondrocytes, which are responsible of the deposition of this specialized ECM, represents a milestone in the regeneration of native articular cartilage. In this study, we fabricated fused deposition modelled (FDM) scaffolds with different pore sizes and architectures from an elastic and biodegradable poly(ester)urethane (PEU) with mechanical properties that can be modulated by design, and that ranged the elasticity of articular cartilage. Cell culture in additive manufactured 3D scaffolds exceeded the chondrogenic potential of the gold-standard pellet culture. In-vitro cell culture studies demonstrated the intrinsic potential of elastic (PEU) to drive the re-differentiation of de-differentiated chondrocytes when cultured in-vitro, in differentiation or basal media, better than pellet cultures. The formation of neo-tissue was assessed as a high deposition of GAGs and fibrillar collagen II, and a high expression of typical chondrogenic markers. Moreover, the collagen II / collagen I ratio commonly used to evaluate the differentiation state of chondrocytes (ratio > 1 being chondrocytes and, ratio < 0 being de-differentiated chondrocytes) was higher than 5. STATEMENT OF SIGNIFICANCE: Tissue engineering of articular cartilage requires material scaffolds capable of driving the deposition of a coherent and specific ECM representative of articular cartilage. Materials explored so far account for low mechanical properties (hydrogels), or are too stiff to mimic the elasticity of the native tissue (traditional polyesters). Here, we fabricated 3D fibrous scaffolds via FDM with a biodegradable poly(ester)urethane. The compressive Young`s modulus and elastic limit of the scaffolds can be tuned by designed, mimicking those of the native tissue. The designed scaffolds showed an intrinsic potential to drive the formation of a GAG and collagen II rich ECM, and to drive a stable chondrogenic cell phenotype.

Elastic materials for tissue engineering applications: Natural, synthetic, and hybrid polymers.

Elastin and collagen are the two main components of elastic tissues and provide the tissue with elasticity and mechanical strength, respectively. Whereas collagen is adequately produced in vitro, production of elastin in tissue-engineered constructs is often inadequate when engineering elastic tissues. Therefore, elasticity has to be artificially introduced into tissue-engineered scaffolds. The elasticity of scaffold materials can be attributed to either natural sources, when native elastin or recombinant techniques are used to provide natural polymers, or synthetic sources, when polymers are synthesized. While synthetic elastomers often lack the biocompatibility needed for tissue engineering applications, the production of natural materials in adequate amounts or with proper mechanical strength remains a challenge. However, combining natural and synthetic materials to create hybrid components could overcome these issues. This review explains the synthesis, mechanical properties, and structure of native elastin as well as the theories on how this extracellular matrix component provides elasticity in vivo. Furthermore, current methods, ranging from proteins and synthetic polymers to hybrid structures that are being investigated for providing elasticity to tissue engineering constructs, are comprehensively discussed. STATEMENT OF SIGNIFICANCE: Tissue engineered scaffolds are being developed as treatment options for malfunctioning tissues throughout the body. It is essential that the scaffold is a close mimic of the native tissue with regards to both mechanical and biological functionalities. Therefore, the production of elastic scaffolds is of key importance to fabricate tissue engineered scaffolds of the elastic tissues such as heart valves and blood vessels. Combining naturally derived and synthetic materials to reach this goal proves to be an interesting area where a highly tunable material that unites mechanical and biological functionalities can be obtained.

Label-Free Detection of Small Organic Molecules by Molecularly Imprinted Polymer Functionalized Thermocouples: Toward In Vivo Applications.

Molecularly imprinted polymers (MIPs), synthetic polymeric receptors, have been combined successfully with thermal transducers for the detection of small molecules in recent years. However, up until now they have been combined with planar electrodes which limits their use for in vivo applications. In this work, a new biosensor platform is developed by roll-coating MIP particles onto thermocouples, functionalized with polylactic acid (PLLA). As a first proof-of-principle, MIPs for the neurotransmitter dopamine were incorporated into PLLA-coated thermocouples. The response of the synthetic receptor layer to an increasing concentration of dopamine in buffer was analyzed using a homemade heat-transfer setup. Binding of the template to the MIP layer blocks the heat transport through the thermocouple, leading to less heat loss to the environment and an overall higher temperature in the measuring chamber. The measured temperature increase is correlated to the neurotransmitter concentration, which enables measurement of dopamine levels in the micromolar regime. To demonstrate the general applicability of the proposed biosensor platform, thermocouples were functionalized with similar MIPs for cortisol and serotonin, indicating a similar response and limit-of-detection. As the platform does not require planar electrodes, it can easily be integrated in, e.g., a catheter. In this way, it is an excellent fit for the current niche in the market of therapeutics and diagnostics. Moreover, the use of a biocompatible and disposable PLLA-layer further illustrates its potential for in vivo diagnostics.

Erasing conformational limitations in N,N'-1,4-butanediyl-bis(6-hydroxy-hexanamide) crystallization from the superheated state of water.

Aliphatic polyamides consist of regularly distributed amide moieties located in an aliphatic chain, off which the segment length can be varied. The crystallization and hence the eventual performance of the material can be tailored by changing the aliphatic lengths, and thus the hydrogen bonding density and the directional chemical positioning of the amide motifs. In this paper, N,N'-1,4-butanediyl-bis(6-hydroxy-hexanamide) crystallized either from the melt or from the superheated state of water is investigated. A comparison with N,N'-1,2-ethanediyl-1,2-bis(6-hydroxy-hexanamide) reveals the role of the hydrogen bonding density on the accommodation of water molecules in amide based crystals grown from the superheated state of water. However, wide-angle X-ray diffraction (WAXD), Fourier transform infrared (FTIR), and solid state 1H and 13C NMR spectroscopy reveal hydrogen bonding between the amide planes, while aliphatic polyamides and N,N'-1,2-diethyl-bis(6-hydroxy-hexanamide) feature hydrogen bonds that reside within the amide plane only. As a consequence, the role of N,N'-1,4-butanediyl-bis(6-hydroxy-hexanamide) crystals as a model system for polyamide 4Y polymers is questionable. However, the thermodynamic and structural behavior as function of temperature is determined by a balance between thermally introduced gauche conformers and hydrogen bonding efficiencies. These crystals enable a thorough investigation in the effect of superheated water on the crystallization of these uniquely hydrogen bonded molecules. Crystallization from the superheated state of water results in denser molecular packing and enhanced hydrogen bonding efficiencies. The induced spatial confinement hinders molecular motion upon heating, and thermodynamically more stable crystals are observed. Although the amide-hydroxyl hydrogen bonded crystals do not favor the accommodation of physically bound water molecules in the lattice, saturation of the amide motifs during crystallization erases conformational restrictions of the planar amide moieties that facilitates maximum hydrogen bonding efficiencies in the eventual lattice.

Interaction of water with N,N'-1,2-ethanediyl-bis(6-hydroxy-hexanamide) crystals: a simulation study.

Recently it has been shown, using a variety of experimental techniques, that water can be hosted in N,N'-1,2-ethanediyl-bis(6-hydroxy-hexanamide) crystals. It forms stable interactions with the hydroxyl groups at the ends of the molecule, as well as with the amide groups. However, with experimental techniques one can not observe the exact hydrogen bonding geometries of the physically bound water molecules. Here a series of molecular dynamics simulations is presented that provide an atomistically detailed picture of the interactions of water with different parts of the crystals.