Functionalizing silk hydrogels with nanoparticles and fibres

This research explores the development of composite silk hydrogels by incorporating different types of silk nanoparticles and microfibres, including Antheraea mylitta (Tasar) silk. The aim was to generate hydrogel composites to ultimately modulate the mechanical properties of Bombyx mori (B. mori) silk hydrogels and enhance cell attachment. B. mori silk lacks the arginine-glycine-aspartic acid (RGD) sequence that is used for cell adhesion. Therefore, introducing RGD-containing Tasar silk within B. mori hydrogels is particularly interesting. This thesis investigated the mechanical properties of silk hydrogels containing various nanoparticles including silica nanoparticles (Chapter 2 and Chapter 3), B. mori and Tasar silk nanoparticles (Chapter 3) as well as silk microfibres . These hydrogel composites were subjected to cell adhesion studies, using DU-145 cells and induced pluripotent stem cell-derived MSCs (iPSCs-MSCs). The research found that ilk hydrogels loaded with 5% w/v silica nanoparticles exhibited higher stiffness than those with lower concentrations (Chapter 2). In Chapter 3, the results showed that silk hydrogels functionalised with nanoparticles had similar stiffness but with variations in stress relaxation while maintaining consistent cell attachment. Silk hydrogels inforced with B. mori and Tasar silk fibres enhanced short-term cell proliferation and attachment, with Tasar silk microfibres being particularly effective. However, cell attachment on silk hydrogels was still less than on tissue culture plastic. Overall, this thesis generated composite silk hydrogels using a spectrum of nanoparticles and silk fibres that in turn modulated the mechanical properties and especially those hydrogels reinforced with silk microfibres, promote short-term cell growth and adhesion.This research explores the development of composite silk hydrogels by incorporating different types of silk nanoparticles and microfibres, including Antheraea mylitta (Tasar) silk. The aim was to generate hydrogel composites to ultimately modulate the mechanical properties of Bombyx mori (B. mori) silk hydrogels and enhance cell attachment. B. mori silk lacks the arginine-glycine-aspartic acid (RGD) sequence that is used for cell adhesion. Therefore, introducing RGD-containing Tasar silk within B. mori hydrogels is particularly interesting. This thesis investigated the mechanical properties of silk hydrogels containing various nanoparticles including silica nanoparticles (Chapter 2 and Chapter 3), B. mori and Tasar silk nanoparticles (Chapter 3) as well as silk microfibres . These hydrogel composites were subjected to cell adhesion studies, using DU-145 cells and induced pluripotent stem cell-derived MSCs (iPSCs-MSCs). The research found that ilk hydrogels loaded with 5% w/v silica nanoparticles exhibited higher stiffness than those with lower concentrations (Chapter 2). In Chapter 3, the results showed that silk hydrogels functionalised with nanoparticles had similar stiffness but with variations in stress relaxation while maintaining consistent cell attachment. Silk hydrogels inforced with B. mori and Tasar silk fibres enhanced short-term cell proliferation and attachment, with Tasar silk microfibres being particularly effective. However, cell attachment on silk hydrogels was still less than on tissue culture plastic. Overall, this thesis generated composite silk hydrogels using a spectrum of nanoparticles and silk fibres that in turn modulated the mechanical properties and especially those hydrogels reinforced with silk microfibres, promote short-term cell growth and adhesion

Microfibre-functionalised silk hydrogels

Silk hydrogels have shown potential for tissue engineering applications, but several gaps and challenges, such as a restricted ability to form hydrogels with tuned mechanics and structural features, still limit their utilisation. Here, Bombyx mori and Antheraea mylitta (Tasar) silk microfibres were embedded within self-assembling B. mori silk hydrogels to modify the bulk hydrogel mechanical properties. This approach is particularly attractive because it creates structured silk hydrogels. First, B. mori and Tasar microfibres were prepared with lengths between 250 and 500 μm. Secondary structure analyses showed high beta-sheet contents of 61% and 63% for B. mori and Tasar microfibres, respectively. Mixing either microfibre type, at either 2% or 10% (w/v) concentrations, into 3% (w/v) silk solutions during the solution–gel transition increased the initial stiffness of the resulting silk hydrogels, with the 10% (w/v) addition giving a greater increase. Microfibre addition also altered hydrogel stress relaxation, with the fastest stress relaxation observed with a rank order of 2% (w/v) > 10% (w/v) > unmodified hydrogels for either fibre type, although B. mori fibres showed a greater effect. The resulting data sets are interesting because they suggest that the presence of microfibres provided potential ‘flow points’ within these hydrogels. Assessment of the biological responses by monitoring cell attachment onto these two-dimensional hydrogel substrates revealed greater numbers of human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) attached to the hydrogels containing 10% (w/v) B. mori microfibres as well as 2% (w/v) and 10% (w/v) Tasar microfibres at 24 h after seeding. Cytoskeleton staining revealed a more elongated and stretched morphology for the cells growing on hydrogels containing Tasar microfibres. Overall, these findings illustrate that hydrogel stiffness, stress relaxation and the iPSC-MSC responses towards silk hydrogels can be tuned using microfibres

Correction : Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly

Correction for ‘Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly’ by Saphia A. L. Matthew et al., RSC Adv., 2022, 12, 7357–7373. https://doi.org/10.2039/D1RA07764C. The authors regret that there were sub-figure placement errors present in Fig. 4 and 5 of the main article. The sub-figure placement error in Fig. 4 was carried into Fig. S3, which shows additional statistical significances. The corrected figures are shown below

Functionalising silk hydrogels with hetero- and homotypic nanoparticles

Despite many reports detailing silk hydrogels, the development of composite silk hydrogels with homotypic and heterotypic silk nanoparticles and their impact on material mechanics and biology have remained largely unexplored. We hypothesise that the inclusion of nanoparticles into silk-based hydrogels enables the formation of homotropic and heterotropic material assemblies. The aim was to explore how well these systems allow tuning of mechanics and cell adhesion to ultimately control the cell–material interface. We utilised nonporous silica nanoparticles as a standard reference and compared them to nanoparticles derived from Bombyx mori silk and Antheraea mylitta (tasar) silk (approximately 100–150 nm in size). Initially, physically cross-linked B. mori silk hydrogels were prepared containing silica, B. mori silk nanoparticles, or tasar silk nanoparticles at concentrations of either 0.05% or 0.5% (w/v). The initial modulus (stiffness) of these nanoparticle-functionalised silk hydrogels was similar. Stress relaxation was substantially faster for nanoparticle-modified silk hydrogels than for unmodified control hydrogels. Increasing the concentrations of B. mori silk and silica nanoparticles slowed stress relaxation, while the opposite trend was observed for hydrogels modified with tasar nanoparticles. Cell attachment was similar for all hydrogels, but proliferation during the initial 24 h was significantly improved with the nanoparticle-modified hydrogels. Overall, this study demonstrates the manufacture and utilisation of homotropic and heterotropic silk hydrogels

Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly

Tuning silk fibroin nanoparticle morphology using nanoprecipitation for bottom-up manufacture is an unexplored field that has the potential to improve particle performance characteristics. The aim of this work was to use both semi-batch bulk mixing and micro-mixing to modulate silk nanoparticle morphology by controlling the supersaturation and shear rate during nanoprecipitation. At flow rates where the shear rate was below the critical shear rate for silk, increasing the concentration of silk in both bulk and micro-mixing processes resulted in particle populations of increased sphericity, lower size, and lower polydispersity index. At high flow rates, where the critical shear rate was exceeded, the increased supersaturation with increasing concentration was counteracted by increased rates of shear-induced assembly. The morphology could be tuned from rod-like to spherical assemblies by increasing supersaturation of the high-shear micro-mixing process, thereby supporting a role for fast mixing in the production of narrow-polydispersity silk nanoparticles. This work provides new insight into the effects of shear during nanoprecipitation and provides a framework for scalable manufacture of spherical and rod-like silk nanoparticles

Smart silk origami as eco-sensors for environmental pollution

Origami folding is an easy, cost-effective, and scalable fabrication method for changing a flat material into a complex 3D functional shape. Here, we created semicrystalline silk films doped with iron oxide particles by mold casting and annealing. The flat silk films could be loaded with natural dyes and folded into 3D geometries using origami principles following plasticization. They performed locomotion under a magnetic field, were reusable and displayed colorimetric stability for 31 days at room temperature in vacuo. The critical parameters for the design of the semi-autonomous silk film, including ease of folding, shape preservation and locomotion in the presence of a magnetic field, were characterized, and pH detection was achieved by eye and by digital image colorimetry with a response time below 1 min. We demonstrate a practical application—a battery-free origami silk boat—as a colorimetric sensor for waterborne pollutants which was reusable at least five times. This work introduces silk eco-sensors and merges responsive actuation and origami techniques