Fabricating and characterising silk powder for biomedical and sorption applications

This research developed a milling technology for ultrafine silk particles and designed novel biocompatible and biodegradable silk composites for repairing hard tissue defects. It also demonstrated high and rapid reversible ion binding properties of silk particles and thereby opened up their application opportunities as advanced green sorbents.

Silk powder: production, characterisation, and application

 For the first time, produced silk particles with unique properties in the range of 7 µm – 200 nm using milling process which opens up new opportunities for silk particle for diverse applications especially drug delivery and tissue scaffolds.

Silk powder for regenerative medicine

Two approaches are used for silk particle production: bottom up and top down. In the bottom up approach, different liquid-solid phase transfer techniques are adapted to fabricate particles from silk solution. In the top down approach, silk fibres are milled by various means to prepare ultrafine silk particles. Many important properties of particles such as size, geometry, porosity, stability and biodegradability are dependent on the specific methods of particle production. These properties influence drug loading and release, delivery modes, biocompatibility and their clearance from the body. Particle properties also determine biomechanical properties of particle reinforced composite scaffolds. Thus correlation between preparation, characterisation and application of silk particles for a specific biomedical application is critical. Progress made in this direction and challenges ahead are discussed in this chapter. © 2014 Woodhead Publishing Limited. All rights reserved.

Fabrication of ultrafine powder from eri silk through attritor and jet milling

Fibroin protein derived from silk fibres has been extensively studied with exciting outcomes for a number of potential advanced biomaterial applications. However, one of the major challenges in applications lies in engineering fibroin into a  desired form using a convenient production technology. In this paper, fabrication of ultrafine powder from eri silk is reported. The silk cocoons were degummed and the extracted silk fibres were then chopped into snippets prior to attritor and air jet milling. Effects of process control agents, material load and material to water ratio during attritor milling were studied. Compared to dry and dry–wet attritor milling, wet process emerged as the preferred option as it caused less colour change and facilitated easy handling. Ultrafine silk powder with a volume based particle size d(0.5) of around 700 nm could be prepared following the sequence of chopping ➔ wet attritor milling ➔ spray drying ➔ air jet milling. Unlike most reported powder production methods, this method could fabricate silk particles in a short time without any pre-treatment on degummed fibre. Moreover, the size range obtained is much smaller than that previously produced using standard milling devices. Reduction in fibre tenacity either shortened the milling time even further or helped bypassing media milling to produce fine powder directly through jet milling. However, such reduction in fibre strength did not help in increasing the ultimate particle fineness. The study also revealed that particle density and particle morphology could be manipulated through appropriate changes in the degumming process.

Graphical Abstract:  Fabrication of eri silk powder using attritor and jet milling is reported. Volume based particle size d(0.5) of around 700 nm could be prepared following the sequence chopping ➔ wet attritor milling ➔ spray drying ➔ air jet milling. No pre-treatments were used and the particle size range obtained is much smaller than that previously produced using standard milling devices. Particle density morphology could be manipulated through appropriate changes of cocoon degumming conditions.

Comparison of milling and solution approach for production of silk particles

Silk particles were produced by regenerating from silk solution, and using a milling method. In the regenerated silk particle production, two methods which are reported to render submicron silk particles were selected. Their particle sizes and structures were compared with particles of milling method already developed by us. The volume median average particle sizes (d(0.5)) of regenerated particles were much higher than what was reported previously. In contrast, milling method could produce particles with adjustable particle sizes ranging from micron to submicron level. All the milled particles had advantage of at least 15. °C higher thermal decomposition temperature than regenerated particles. They had silk II structure, and the crystallinity reduced as particle fineness increased, but remained higher than regenerated particles of similar sizes. © 2014 Elsevier B.V.

Drug loading and release studies for milled silk particles of different sizes

Milled silk particles with volume median particle size (d(0.5)) of 7 μm and 281 nm as well as silk snippets were used for loading of model drugs Orange G, Azophloxine, Rhodamine B, and Crystal Violet. Loading and release of these chemicals depended on the size of silk particles, pH, and the structure and properties of model drugs. Both types of silk particles reached equilibrium loading in less than 10 min due to high surface area whereas silk fibres needed more than 2-3 days to reach equilibrium, depending on the drug type. The uptake rate in fibres could be improved by increasing temperature. Both fibres and particles could slowly release the drugs over many days at 37 °C without a significant initial burst. As particle size decreased, the amount of model drug release also decreased. The release of drugs by the silk fibres was quicker than the silk particles.

An investigation into transition metal ion binding properties of silk fibers and particles using radioisotopes

Silk is a structural protein fiber that is stable over a wide pH range making it attractive for use in medical and environmental applications. Variation in amino acid composition has the potential for selective binding for ions under varying conditions. Here we report on the metal ion separation potential of Mulberry and Eri silk fibers and powders over a range of pH. Highly sensitive radiotracer probes, ⁶⁴Cu²⁺, ¹⁰⁹Cd²⁺, and ⁵⁷Co²⁺ were used to study the absorption of their respective stable metal ions Cu²⁺, Cd²⁺, and Co²⁺ into and from the silk sorbents. The total amount of each metal ion absorbed and time taken to reach equilibrium occurred in the following order: Cu²⁺ > Cd²⁺ > Co ²⁺. In all cases the silk powders absorbed metal ions faster than their respective silk fibers. Intensive degumming of the fibers and powders significantly reduced the time to absorb respective metal ions and the time to reach equilibrium was reduced from hours to 5-15 min at pH 8. Once bound, 45-100% of the metal ions were released from the sorbents after exposure to pH 3 buffer for 30 min. The transition metal ion loading capacity for the silk sorbents was considerably higher than that found for commercial ion exchange resins (AG MP-50 and AG 50W-X2) under similar conditions. Interestingly, total Cu²⁺ bound was found to be higher than theoretically predicted values based on known specific Cu²⁺ binding sites (AHGGYSGY), suggesting that additional (new) sites for transition metal ion binding sites are present in silk fibers.

Recent innovations in silk biomaterials

Silk contains a fibre forming protein, fibroin, which is biocompatible, particularly after removing the potentially immunogenic non-fibroin proteins. Silk can be engineered into a wide range of materials with diverse morphologies. Moreover, it is possible to regenerate fibroin with a desired amount of crystallinity, so that the biodegradation of silk materials can be controlled. These advantages have sparked new interest in the use of silk fibroin for biomedical applications, including tissue engineering scaffolds and carriers for sustained release of biologically active molecules. This article summarizes the current research related to the formation of silk materials with different morphologies, their biocompatibility, and examples of their biomedical applications. Recent work on the preparation of silk particles by mechanical milling and their applications in silk composite scaffolds is also discussed.

Protein fibre powder and new applications

Protein fibers such as silk and wool have been used as textile fibers for centuries. It is only in recent years that these fibers have been converted into fine powder forms for non-textile applications. This presentation will cover our recent research in protein fiber powders. Ultra-fine powders from different protein fibers have been produced using a combination of media and non media milling techniques. New application examples of these fine powders are discussed. These applications include hybrid fibers combining the advantages of natural and synthetic polymer fibers, tissue engineering composite scaffolds with enhanced biomechanical properties, and metal ion absorption.

Relationship between processing, surface energy and bulk properties of ultrafine silk particles

Silk particles of different sizes and shapes were produced by milling and interactions with a series of polar and non-polar gaseous probes were investigated using an inverse gas chromatography technique. The surface energy of all silk materials is mostly determined by long range dispersive interactions such as van der Waals forces. The surface energy increases and surface energy heterogeneity widens after milling. All samples have amphoteric surfaces and the concentration of acidic groups increases after milling while the surfaces remain predominantly basic. We also examined powder compression and flow behaviours using a rheometer. Increase in surface energy, surface area, and static charges in sub-micron air jet milled particles contributed to their aggregation and therefore improved flowability. However they collapse under large pressures and form highly cohesive powder. Alkaline hydrolysis resulted in more crystalline fibres which on milling produced particles with higher density, lower surface energy and improved flowability. The compressibility, bulk density and cohesion of the powders depend on the surface energy as well as on particle size, surface area, aggregation state and the testing conditions, notably the consolidated and unconsolidated states. The study has helped in understanding how surface energy and flowability of particles can be changed via different fabrication approaches.

Structure and characteristics of milled silk particles

This study examined the structure, thermal property, and ion adsorption of silk particles. The particles were prepared by attritor-bead mill combination, using alkaline (pH10) charge repulsion and surfactant steric repulsion methods. Both methods produced particles with a dominant β-sheet structure, similar to the silk fibre. There was no significant difference in the decomposition temperatures for either the silk fibre or the micro/nano silk particles. An important finding from this study is clear evidence of reduction of amorphous content during the final stage of powdering using the bead mill. As a result, despite reduction in β-sheet crystallites with the progressive milling, the relative β-sheet content actually increased during this process. However, intermolecular forces between the β-sheets reduced significantly and hence the XRD results showed significant reduction in crystallinity in nano silk particles but crystal forming segments remained with β-sheet conformations after milling. The structural change influenced the ion-adsorption property where particle-size reduction resulted in a significant increase in both the rate and volume of HCrO4- adsorption. © 2014 Elsevier B.V.

Micro-porous nickel produced by powder metallurgy

Micro-porous nickel (Ni) with an open cell structure was fabricated by a special powder metallurgical process, which includes the adding of a space-holding material. The average pore size of the micro-porous Ni samples approximated 30 μm and 150 μm, and the porosity ranged from 60 % to 80 %. The porous characteristics of the Ni samples were observed using scanning electron microscopy (SEM) and the mechanical properties were evaluated using compressive tests. For comparison, porous Ni samples with a macro-porous structure prepared by both powder metallurgy
(pore size 800 μm) and the traditional chemical vapour deposition (CVD) method (pore size 1300 μm) were also presented. Results indicated that the porous Ni samples with a micro-porous structure exhibited different deformation behaviour and dramatically increased mechanical properties,
compared to those of the macro-porous Ni samples.