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A variety of artificial silk spinning approaches are attempted to mimic the natural spinning process found in silkworms and spiders, yet instantaneous silk fiber formation with hierarchical structure under physiological and ambient conditions without post‐treatment procedures remains unaddressed. Here, this work reports a new strategy to fabricate silk protein‐based aerosols and silk fibers instantaneously in situ using a spray device, avoiding complicated and costly advanced manufacturing techniques. The key to success is the instantaneous conformational transition of silk fibroin from random coil to β‐sheet right before spraying by mixing silk and polyethylene glycol (PEG) solutions in the spray device, allowing aerosols and silk fibers to be sprayed, with further control achieved via the molecular weight of silk. The spinning process of the spray device is based on the use of green solvents, that is, all steps of instant conformational transition of silk fibroin are carried out in aqueous conditions or with buffers at ambient conditions, in combination with shear and elongational flow caused by the hydraulic pressure generated in the spray container. The system supports a portable and user‐friendly system that could be used for drug delivery carriers, wound coating materials, and rapid silk fiber conformal coatings on surfaces. [ABSTRACT FROM AUTHOR]

The physical and mechanical properties of structural proteins such as silk fibroin can be modified by controlled conformational change, which is regularly monitored by Fourier transform infrared spectroscopy by peak fitting of the amide I band envelope. Although many variables affecting peak shape are well established, there is no fixed methodology to compare and follow secondary structural differences without significant operator input especially where low frequency spectral noise is a problem. The aim of this contribution is to establish a method for such analyses to be carried at high levels of autonomy to prevent subjective or erroneous fitting. A range of approaches was trialled with optimal peak parameters selected based on overall goodness of fit and reproducibility of fit of replicate sample spectra. The method was successfully tested against reference proteins having contrasting β content and the rationale for parameter selection is presented. Further, we applied this method to measure the effect of conformational change on the energy of the amide I band of silk fibroin during annealing. Energy changes were ca. 400 kJ mol −1 of fibroin. To confirm that this energy change was a consequence of increased hydrogen bonding we used a Thioflavin T staining method typically used to identify β aggregate type structures in amyloid plaques. We propose that the approach described herein can aid in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity. Statement of Significance The physical and mechanical properties of proteins including silk fibroin can be modified by controlled structural change, which is regularly monitored by Fourier transform infrared spectroscopy (FTIR) by peak fitting of the amide I band. Currently there is no fixed methodology to compare and follow secondary structural differences without significant operator input leading to subjectivity and error. This contribution establishes a method for such analyses to be carried at high levels of autonomy applicable to a wide range of proteins and the conformational changes have been quantified as a single energy change output, which clearly shows the progression of the annealing process used. We propose that the approach can help in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity. [ABSTRACT FROM AUTHOR]

Freeform fabrication has emerged as a key direction in printing biologically-relevant materials and structures. With this emerging technology, complex structures with microscale resolution can be created in arbitrary geometries and without the limitations found in traditional bottom-up or top-down additive manufacturing methods. Recent advances in freeform printing have used the physical properties of microparticle-based granular gels as a medium for the submerged extrusion of bioinks. However, most of these techniques require post-processing or crosslinking for the removal of the printed structures (Miller et al., 2015; Jin et al., 2016) [1,2] . In this communication, we introduce a novel method for the one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite) and polyethylene glycol (PEG). Silk fibroin has been used as a biopolymer for bioprinting in several contexts, but chemical or enzymatic additives or bulking agents are needed to stabilize 3D structures. Our method requires no post-processing of printed structures and allows for in situ physical crosslinking of pure aqueous silk fibroin into arbitrary geometries produced through freeform 3D printing. Statement of Significance 3D bioprinting has emerged as a technology that can produce biologically relevant structures in defined geometries with microscale resolution. Techniques for fabrication of free-standing structures by printing into granular gel media has been demonstrated previously, however, these methods require crosslinking agents and post-processing steps on printed structures. Our method utilizes one-step gelation of silk fibroin within a suspension of synthetic nanoclay (Laponite), with no need for additional crosslinking compounds or post processing of the material. This new method allows for in situ physical crosslinking of pure aqueous silk fibroin into defined geometries produced through freeform 3D printing. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogels are the focus of extensive research due to their potential use in fields including biomedical, pharmaceutical, biosensors, and cosmetics. However, the general weak mechanical properties of hydrogels limit their utility. Here, pristine silk fibroin (SF) hydrogels with excellent mechanical properties are generated via a binary‐solvent‐induced conformation transition (BSICT) strategy. In this method, the conformational transition of SF is regulated by moderate binary solvent diffusion and SF/solvent interactions. β‐sheet formation serves as the physical crosslinks that connect disparate protein chains to form continuous 3D hydrogel networks, avoiding complex chemical and/or physical treatments. The Young's modulus of these new BSICT–SF hydrogels can reach up to 6.5 ± 0.2 MPa, tens to hundreds of times higher than that of conventional hydrogels (0.01–0.1 MPa). These new materials fill the “empty soft materials' space” in the elastic modulus/strain Ashby plot. More remarkably, the BSICT–SF hydrogels can be processed into different constructions through different polymer and/or metal‐based processing techniques, such as molding, laser cutting, and machining. Thus, these new hydrogel systems exhibit potential utility in many biomedical and engineering fields. [ABSTRACT FROM AUTHOR]

The production of structural and functional materials with enhanced mechanical properties through the integration of soft and hard components is a common approach to Nature's material design. However, directly mimicking these optimized design routes in the lab for practical applications remains challenging. For example, graphene and silk are two materials with complementary mechanical properties that feature ultrahigh stiffness and toughness, respectively. Yet, no simple and controllable approach is developed to homogeneously integrate these two components into functional composites, mainly due to the hydrophobicity and chemical inertness of graphene. In this study, well-dispersed and highly stable graphene/silk fibroin (SF) suspension systems are developed, which are suitable for processing to fabricate polymorphic materials, such as films, fibers, and coatings. The obtained graphene/SF nanocomposites maintain the electronic advantages of graphene, and they also allow tailorable mechanical performance to form including ultrahigh stretchable (with a strain to failure to 611 ± 85%), or high strength (339 MPa) and high stiffness (7.4 GPa) material systems. More remarkably, the electrical resistances of these graphene/SF materials are sensitive to material deformation, body movement, as well as humidity and chemical environmental changes. These unique features promise their utility as wearable sensors, smart textiles, intelligent skins, and human-machine interfaces. [ABSTRACT FROM AUTHOR]

Various techniques have been employed to entrap fragrant oils within microcapsules or microparticles in the food, pharmaceutical, and chemical industries for improved stability and delivery. In the present work, we describe the use of silk protein microparticles for encapsulating fragrant oils using ambient processing conditions to form an all-natural biocompatible matrix. These microparticles are stabilized via physical crosslinking, requiring no chemical agents, and are prepared with aqueous and ambient processing conditions using polyvinyl alcohol-silk emulsions. The particles were loaded with fragrant oils via direct immersion of the silk particles within an oil bath. The oil-containing microparticles were coated using alternating silk and polyethylene oxide layers to control the release of the oil from the microspheres. Particle morphology and size, oil loading capacity, release rates as well as silk-oil interactions and coating treatments were characterized. Thermal analysis demonstrated that the silk coatings can be tuned to alter both retention and release profiles of the encapsulated fragrance. These oil-containing particles demonstrate the ability to adsorb and controllably release oils, suggesting a range of potential applications including cosmetic and fragrance utility. [ABSTRACT FROM AUTHOR]

Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk–tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly- d -lysine. When patterned by drying on grooved polydimethylsiloxane (3.5 μm groove width, 0.5 μm groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk–tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs. [ABSTRACT FROM AUTHOR]

Cartilage tissue lacks an intrinsic capacity for self-regeneration due to slow matrix turnover, a limited supply of mature chondrocytes and insufficient vasculature. Although cartilage tissue engineering has achieved some success using agarose as a scaffolding material, major challenges of agarose-based cartilage repair, including non-degradability, poor tissue–scaffold integration and limited processing capability, have prompted the search for an alternative biomaterial. In this study, silk fiber–hydrogel composites (SF–silk hydrogels) made from silk microfibers and silk hydrogels were investigated for their potential use as a support material for engineered cartilage. We demonstrated the use of 100% silk-based fiber–hydrogel composite scaffolds for the development of cartilage constructs with properties comparable to those made with agarose. Cartilage constructs with an equilibrium modulus in the native tissue range were fabricated by mimicking the collagen fiber and proteoglycan composite architecture of native cartilage using biocompatible, biodegradable silk fibroin from Bombyx mori . Excellent chondrocyte response was observed on SF–silk hydrogels, and fiber reinforcement resulted in the development of more mechanically robust constructs after 42 days in culture compared to silk hydrogels alone. Thus, we demonstrate the versatility of silk fibroin as a composite scaffolding material for use in cartilage tissue repair to create functional cartilage constructs that overcome the limitations of agarose biomaterials, and provide a much-needed alternative to the agarose standard. [ABSTRACT FROM AUTHOR]

The goal of this study was to determine the impact of silk biomaterial structure (e.g. solution, hydrogel, film) on proteolytic susceptibility. In vitro enzymatic degradation of silk fibroin hydrogels and films was studied using a variety of proteases, including proteinase K, protease XIV, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1) and MMP-2. Hydrogels were used to assess bulk degradation while films were used to assess surface degradation. Weight loss, secondary structure determined by Fourier transform infrared spectroscopy and degradation products analyzed via sodium dodecyl sulfate–polyacrylamide gel electrophoresis were used to evaluate degradation over 5 days. Silk films were significantly degraded by proteinase K, while silk hydrogels were degraded more extensively by protease XIV and proteinase K. Collagenase preferentially degraded the β-sheet content in hydrogels while protease XIV and α-chymotrypsin degraded the amorphous structures. MMP-1 and MMP-2 degraded silk fibroin in solution, resulting in a decrease in peptide fragment sizes over time. The link between primary sequence mapping with protease susceptibility provides insight into the role of secondary structure in impacting proteolytic access by comparing solution vs. solid state proteolytic susceptibility. [ABSTRACT FROM AUTHOR]

Neural engineering provides promise for cell therapy by integrating the host brain with brain-machine-interface technologies in order to externally modulate functions. Long-term interfaces with the host brain remain a critical challenge due to insufficient graft cell survivability and loss of brain electrode sensitivity over time. Here, integrated neuron-electrode interfaces are developed on thin flexible and transparent silk films as brain implants. Mechanical properties and surface topography of silk films are optimized to promote cell survival and alignment of primary rat cortical cells. Compartmentalized neural cultures and co-patterned electrode arrays are incorporated on the silk films with built-in wire connections. Electrical stimulation via electrodes embedded in the films activated surrounding neurons to produce evoked calcium responses. In mice brains, silk film implants show conformal contact capable of modulating host brain cells with minimal inflammatory response and stable indwelling for weeks. The approach of combining cell therapy and brain electrodes could provide sustained functional interfaces with ex vivo control with spatial precision. [ABSTRACT FROM AUTHOR]

Abstract: The fabrication of new dermal substitutes providing mechanical support and cellular cues is urgently needed in dermal reconstruction. Silk fibroin (SF)/chondroitin sulfate (CS)/hyaluronic acid (HA) ternary scaffolds (95–248μm in pore diameter, 88–93% in porosity) were prepared by freeze-drying. By the incorporation of CS and HA with the SF solution, the chemical potential and quantity of free water around ice crystals could be controlled to form smaller pores in the SF/CS/HA ternary scaffold main pores and improve scaffold equilibrium swelling. This feature offers benefits for cell adhesion, survival and proliferation. In vivo SF, SF/HA and SF/CS/HA (80/5/15) scaffolds as dermal equivalents were implanted onto dorsal full-thickness wounds of Sprague–Dawley rats to evaluate wound healing. Compared to SF and SF/HA scaffolds, the SF/CS/HA (80/5/15) scaffolds promoted dermis regeneration, related to improved angiogenesis and collagen deposition. Further, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) expression in the SF/CS/HA (80/5/15) groups were investigated by immunohistochemistry to assess the mechanisms involved in the stimulation of secretion of VEGF, PDGF and bFGF and accumulation of these growth factors related to accelerated wound process. These new three-dimensional ternary scaffolds offer potential for dermal tissue regeneration. [Copyright &y& Elsevier]

Abstract: Silk fibroin is a potent alternative to other biodegradable biopolymers for bone tissue engineering (TE), because of its tunable architecture and mechanical properties, and its demonstrated ability to support bone formation both in vitro and in vivo. In this study, we investigated a range of silk scaffolds for bone TE using human adipose-derived stem cells (hASCs), an attractive cell source for engineering autologous bone grafts. Our goal was to understand the effects of scaffold architecture and biomechanics and use this information to optimize silk scaffolds for bone TE applications. Silk scaffolds were fabricated using different solvents (aqueous vs. hexafluoro-2-propanol (HFIP)), pore sizes (250–500μm vs. 500–1000μm) and structures (lamellar vs. spherical pores). Four types of silk scaffolds combining the properties of interest were systematically compared with respect to bone tissue outcomes, with decellularized trabecular bone (DCB) included as a “gold standard”. The scaffolds were seeded with hASCs and cultured for 7weeks in osteogenic medium. Bone formation was evaluated by cell proliferation and differentiation, matrix production, calcification and mechanical properties. We observed that 400–600μm porous HFIP-derived silk fibroin scaffold demonstrated the best bone tissue formation outcomes, as evidenced by increased bone protein production (osteopontin, collagen type I, bone sialoprotein), enhanced calcium deposition and total bone volume. On a direct comparison basis, alkaline phosphatase activity (AP) at week 2 and new calcium deposition at week 7 were comparable to the cells cultured in DCB. Yet, among the aqueous-based structures, the lamellar architecture induced increased AP activity and demonstrated higher equilibrium modulus than the spherical-pore scaffolds. Based on the collected data, we propose a conceptual model describing the effects of silk scaffold design on bone tissue formation. [Copyright &y& Elsevier]

Abstract: Physical guidance cues have been exploited to stimulate neuron adhesion and neurite outgrowth. In the present study, three-dimensional (3-D) silk fibroin scaffolds with uniaxial multichannels (42–142μm in diameter) were prepared by a directional temperature field freezing technique, followed by lyophilization. By varying the initial silk fibroin concentration, the chemical potential and quantity of free water around cylindrical ice crystals could be controlled to control the cross-section morphology of the scaffold channels. Aligned ridges also formed on the inner surface of the multichannels in parallel to the direction of the channels. In vitro, primary hippocampal neurons were seeded in these 3-D silk fibroin scaffolds with uniaxial multichannels of ∼120μm in diameter. The morphology of the neurons was multipolar and alignment along the scaffold channels was observed. Cell–cell networks and cell–matrix interactions established by newly formed axons were observed after 7days in culture. These neurons expressed β-III-tubulin, nerve filament and microtubule-associated protein, while glial fibrillary acidic protein immunofluorescence was barely above background. The ridges on the inner surface of the channels played a critical role in the adhesion and extension of neurons by providing continuous contact guidance. These new 3-D silk scaffolds with uniaxial multichannels provided a favorable microenvironment for the development of hippocampal neurons by guiding axonal elongation and cell migration. [Copyright &y& Elsevier]

The present study was undertaken to evaluate ectopic new bone formation effects of apatite-coated silk fibroin scaffolds (mSS) seeded with adenovirus-mediated bone morphogenic protein-2 gene (AdBMP-2) transduced canine bone marrow stromal cells (bMSCs) in nude mice. In this study, bMSCs derived from canine were cultured and transduced with AdBMP-2 adenovirus-mediated enhanced green fluorescent protein gene (AdEGFP) in vitro. Osteogenic differentiation of bMSCs was determined by alkaline phosphatase (ALP) activity analysis, and the transcript levels for BMP-2, osteopontin (OPN), osteocalcin (OCN) and bone sialoprotein (BSP) genes via real-time quantitative PCR (RT-qPCR) analysis. The ectopic bone formation effects of mSS seeded with AdBMP-2-modified bMSCs were evaluated through histological and histomorphological analysis 4, 8 and 12 weeks post-operation in nude mice. ALP activity was statistically increased in the AdBMP-2 group, when compared with control groups. The mRNA expression of BMP-2, OPN, OCN and BSP was also statistically up-regulated 6 and 9 days after AdBMP-2 transduction. Significantly higher bone volume was achieved in AdBMP-2-transduced bMSCs/mSS constructs than that of AdEGFP-transduced bMSCs/mSS or bMSCs/mSS groups at 4, 8 and 12 weeks (P < 0.01). These results demonstrated that mSS seeded with AdBMP-2-transduced canine bMSCs can promote ectopic new bone formation and maturation in nude mice, suggesting the potential of this silk-scaffold-based tissue-engineered bone for further bone regeneration studies in canine models. [ABSTRACT FROM AUTHOR]

Abstract: A silk fibroin gel system (e-gel), formed with weak electric fields, has potential utility in medical materials and devices. The mechanism of silk e-gel formation was studied to gain additional insight into the process and control of the material properties. Silk fibroin nanoparticles with sizes of tens of nanometers, composed of metastable conformations, were involved in e-gel formation. Under electric fields the nanoparticles rapidly assembled into larger nano- or microspheres with size range from tens of nanometers to several microns. Repulsive forces from the negative surface charge of the acidic groups on the protein were screened by the local decrease in solution pH in the vicinity of the positive electrode. By controlling the formation and content of silk fibroin nanoparticles e-gels could be formed even from low concentration silk fibroin solutions (1%). When e-gel formation was reversed to the solution state the aggregated nano- and microspheres dispersed into solution, a significant observation related to future applications for this process, such as drug delivery. [Copyright &y& Elsevier]

Silk fibroin in material formats provides robust mechanical properties, and thus is a promising protein for 3D printing inks for a range of applications, including tissue engineering, bioelectronics, and bio-optics. Among the various crosslinking mechanisms, photo-crosslinking is particularly useful for 3D printing with silk fibroin inks due to the rapid kinetics, tunable crosslinking dynamics, light-assisted shape control, and the option to use visible light as a biocompatible processing condition. Multiple photo-crosslinking approaches have been applied to native or chemically modified silk fibroin, including photo-oxidation and free radical methacrylate polymerization. The molecular characteristics of silk fibroin, i.e., conformational polymorphism, provide a unique method for crosslinking and microfabrication via light. The molecular design features of silk fibroin inks and the exploitation of photo-crosslinking mechanisms suggest the exciting potential for meeting many biomedical needs in the future. [ABSTRACT FROM AUTHOR]

[Display omitted] The ability to reverse engineer the silkworm fiber has led to tremendous advancements in the field of silk materials over the last several decades, with silk fibroin materials applied to biomedical, food, and high technology industries. This progress is largely due to the ability to process silk into a regenerated silk fibroin solution that can be engineered into a variety of material formats and stabilized by the reintroduction of the non-covalent native β-sheet protein structure. The next revolution in silk materials involves stabilizing silk fibroin through covalent crosslinking and plasticization. These approaches have transformed silk into a material that is not only strong, but also elastic and flexible, making it compatible with modern fabrication approaches. This has significantly broadened silk material fabrication strategies to include photolithography, digital light processing, and extrusion-based 3D printing. As a result, silk can now be used in a range of applications including ocular prostheses, bio-adhesives, tissue engineering matrices, green biodegradable LEDs and batteries, on-skin and implantable sensors, and bioplastics. In this review, we discuss the evolution of crosslinking in silk materials, focusing on covalent tyrosine- and methacrylate-based crosslinks, and on the structural changes and crosslinking brough about by plasticizing silk using glycerol and calcium ions. We describe how advances in silk crosslinking led to the development of unique materials, paving the way for new fabrication approaches and applications across multiple industries, ushering in a new era of silk materials. [ABSTRACT FROM AUTHOR]

Abstract: The development of biomaterials that mimic the physiological binding of growth factors to the extracellular matrix (ECM) is an appealing strategy for advanced growth factor delivery systems. In vivo, fibroblast growth factor 2 (FGF-2) binds to the sulfated glycosaminoglycan heparan sulfate, which is a major component of the ECM. Therefore, we tested whether silk fibroin (SF) decorated with a sulfonated moiety could mimic the natural ECM environment and lead to advanced delivery of this heparin-binding growth factor. Using a diazonium coupling reaction, modified SF derivatives containing approximately 20, 40, 55 and 70 sulfonic acid groups per SF molecule were obtained. Films of the SF derivative decorated with 70 sulfonic acid groups per SF molecule resulted in a 2-fold increase in FGF-2 binding as compared to native SF. More than 99% of bound FGF-2 could be retained on all SF derivatives. However, protection of FGF-2 potency was only achieved with at least 40 sulfonic acid groups per SF molecule, as observed by reduced metabolic activity and enhanced levels of phosphorylated extracellular signal-regulated kinases (pERK1/2) in cultured human mesenchymal stem cells (hMSCs). This study introduces a first step towards the development of an ECM-mimicking biomaterial for sustained, non-covalent binding, controlled delivery and preserved potency of biomolecules. [Copyright &y& Elsevier]

In situ 3D printing is an emerging technique designed for patient-specific needs and performed directly in the patient's tissues in the operating room. While this technology has progressed rapidly, several improvements are needed to push it forward for widespread utility, including ink formulations and optimization for in situ context. Silk fibroin inks emerge as a viable option due to the diverse range of formulations, aqueous processability, robust and tunable mechanical properties, and self-assembly via biophysical adsorption to avoid exogenous chemical or photochemical sensitizer additives, among other features. In this review, we focus on this new frontier of 3D in situ printing for tissue regeneration, where silk is proposed as candidate biomaterial ink due to the unique and useful properties of this protein polymer. In vitro 3D printing techniques have challenges that limit their clinical translation, including multistep processes, mismatches with patient-specific defects, risk of contamination, and postprocessing manipulation requirements. In situ 3D printing, the next frontier for 3D printing, aims to fabricate new tissues and organs in vivo , in the surgical setting, directly in the patient. Inks remain a challenge for this transition to in situ 3D printing, requiring fast gelation, high shape fidelity, minimal if any postprocessing, robust mechanical properties tunable to the target tissue, and biocompatibility. Versatile and appropriate inks, such as those developed from silk fibroin, offer a foundation for this translation, based on their unique amphiphilic structure, versatility in physical crosslinking, mechanical properties, biocompatibility, and tunable degradation. [ABSTRACT FROM AUTHOR]

Poly(d , l -lactide- co -glycolide) (PLGA) microspheres have been used as an injectable depot for prolonged release of octreotide (Sandostatin LAR®), a peptide drug for the treatment of acromegaly and gastrointestinal tumors. However, acylation and incomplete release of the encapsulated octreotide, as well as acidic degradation product-induced inflammation are the major challenges hampering widespread clinical applications of this delivery system. The purpose of this study was to develop a novel octreotide-delivering system utilizing naturally derived biodegradable material, silk fibroin (SF). Octreotide acetate was encapsulated in the SF microspheres with a high loading (8–10 wt%) using polyethylene glycol (PEG)-assisted emulsification method. The octreotide-SF microspheres exhibited a silk I structure (low crystallinity) and burst release in in vitro release studies. Ethanol treatment after microsphere formation significantly increased β-sheet and silk II structure (high crystallinity) of the microspheres, significantly reducing the burst release and resulting in zero-order sustained release of octreotide over 102 days, and the data could be fit to the diffusion-driven release model. After the ethanol-treated microspheres were intramuscularly injected into rats at low (2 mg/kg) and high (8 mg/kg) octreotide doses, the plasma concentration of octreotide in the high dose group remained high (>50 pg/mL) at day 28 when compared to that of the control (pure drug at low dose) and low dose microsphere group. Interestingly, the plasma concentration for the high dose group at day 56 dramatically increased to >280 pg/mL observed at day 28. The low dose microsphere group showed a similar increase, but at a much lower level. The rebound octreotide level likely reflected degradation of the SF matrix which released tightly bound/trapped octreotide. Therefore, SF microspheres can deliver octreotide over a long period of time with release kinetics and the mechanism different from PLGA microsphere system. • Silk fibroin microspheres were fabricated using a novel PEG-assisted emulsification-condensation approach • A peptide drug, octreotide, was encapsulated in the microspheres at high loading and encapsulation efficiency • More than 100 days in vitro release under the sink condition was achieved successfully • Octreotide was released biphasically to blood for more than 56 days after a single-dose intramuscular injection in rats [ABSTRACT FROM AUTHOR]

Abstract Injectable silk hydrogels are ideal carriers of therapeutic agents due to their biocompatibility and low immunogenicity. Injectable silk hydrogels for bone regeneration have been previously developed but often utilize expensive biologics. In this study, we have developed an injectable silk composite incorporated with a triphasic ceramic called MSM-10 (54 Mg 2 SiO 4 , 36 Si 3 Sr 5 and 10 MgO (wt%)) capable of simultaneously releasing magnesium, silicon, and strontium ions into its environment. These ions have been previously reported to possess therapeutic effects for bone regeneration. MSM-10 particles were incorporated into the silk hydrogels at various weight percentages [0.1 (SMH-0.1), 0.6 (SMH-0.6), 1 (SMH-1) and 2 (SMH-2)]. The effects of the released ions on the physicochemical and biological properties of the silk hydrogel were comprehensively evaluated. Increased MSM-10 loading was found to hinder the gelation kinetics of the silk hydrogel through the reduction of beta-sheet phase formation, which in turn affected the required sonication time for gelation, compressive strength, force of injection, microstructure and in vitro degradation rate. Primary human osteoblasts seeded on SMH-0.6 demonstrated increased proliferation and early alkaline phosphatase activity, as well as enhanced osteogenic gene expression compared to pure silk hydrogel and SMH-0.1. In vivo results in subcutaneous mouse models showed both decreased fibrous capsule formation and increased number of new blood vessels around the injected SMH-0.1 and SMH-0.6 implants compared to pure silk hydrogels. The results in this study indicate that the ions released from MSM-10 is able to influence the physicochemical and biological properties of silk hydrogels, and SMH-0.6 in particular shows promising properties for bone regeneration. Highlights • Silk hydrogels able to release bioactive Mg, Si and Sr ions were prepared by sonication and with novel ceramic particulates. • The release of Mg, Si and Sr ions markedly improved in vitro osteogenesis of silk hydrogels. • Incorporation of ceramic particulate changed physical, chemical and mechanical properties of silk hydrogels. • Modified hydrogels provided better environment for angiogenesis and reduced surrounding fibrous capsule formation in vivo. [ABSTRACT FROM AUTHOR]

Silk fibers spun by silkworms and spiders exhibit exceptional mechanical properties with a unique combination of strength, extensibility and toughness. In contrast, the mechanical properties of regenerated silk materials can be tuned through control of the fabrication process. Here we introduce a biomimetic, all-aqueous process, to obtain bulk regenerated silk-based materials for the fabrication of functionalized orthopedic devices. The silk materials generated in the process replicate the nano-scale structure of natural silk fibers and possess excellent mechanical properties. The biomimetic materials demonstrate excellent machinability, providing a path towards the fabrication of a new family of resorbable orthopedic devices where organic solvents are avoided, thus allowing functionalization with bioactive molecules to promote bone remodeling and integration. [ABSTRACT FROM AUTHOR]

Silk fibroin is a high molecular weight amphiphilic protein that self-assembles into robust biomaterials with remarkable properties including stabilization of biologicals and tunable release kinetics correlated to processing conditions. Cells, antibiotics, monoclonal antibodies and peptides, among other biologics, have been encapsulated in silk using various processing approaches and material formats. The mechanistic basis for the entrapment and stabilization features, along with insights into the modulation of release of the entrained compounds from silks will be reviewed with a focus on stabilization of bioactive molecules. [ABSTRACT FROM AUTHOR]

A silk fibroin (silk) hydrogel was prepared by using diglycidyl ether (BDDE), a chemical crosslinker commonly used to generate Food and Drug Administration (FDA)-approved hyaluronic acid (HA) medical products. The silk/BDDE hydrogels exhibited high elasticity (compressive modulus of 166 ± 15.0 kPa), anti-fatigue properties, and stable structure and mechanical strength in aqueous solution. Chemical crosslinking was conducted in a high concentration (9.3 M) of lithium bromide (LiBr) solution, a salt that is commonly used to dissolve degummed silk fibers during silk solubilization. The unfolded and extended structure of silk molecules with these reaction conditions, as well as the unique ionic environment provided by LiBr facilitated a high degree of crosslinking in the hydrogel. Similar hydrogels were not obtained when the silk was dissolved in other silk fiber-dissolving reagents (e.g., Ajisawa's, formic acid (FA)/LiBr, FA/CaCl 2 solutions), likely because partially folded silk structures and the ionic conditions with these reagents were less favorable for the crosslinking reaction. Based on these findings, silk/BDDE hydrogel spheres were prepared using an oil/water (o/w) emulsification method and biocompatibility and biodegradation were evaluated in vivo , along with other silk gel control systems (e.g., enzyme-catalyzed di-tyrosine and pulverized silk/BDDE gel particles with irregular shapes). Histological and immunohistochemical analyses demonstrated that the silk/BDDE hydrogel spheres were biocompatible and served as a bio-lubricant to treat osteoarthritis (OA). The intra-articular injection of the gel spheres reduced pain as measured with OA rats, reduced cartilage damage and resisted the digestive environment in the articular cavity for extended time frames (>4 weeks), suggesting utility for pain relief and sustained drug release for future OA treatments. [ABSTRACT FROM AUTHOR]

Surgical management of long-gap esophageal defects with autologous gastrointestinal tissues is frequently associated with adverse complications including organ dysmotility, dysphagia, and donor site morbidity. In order to develop alternative graft options, bi-layer silk fibroin (SF) scaffolds were investigated for their potential to support functional tissue regeneration in a rodent model of esophageal repair. Onlay esophagoplasty was performed with SF matrices (N = 40) in adult rats for up to 2 m of implantation. Parallel groups consisted of animals implanted with small intestinal submucosa (SIS) scaffolds (N = 22) or sham controls receiving esophagotomy alone (N = 20). Sham controls exhibited a 100% survival rate while rats implanted with SF and SIS scaffolds displayed respective survival rates of 93% and 91% prior to scheduled euthanasia. Animals in each experimental group were capable of solid food consumption following a 3 d post-op liquid diet and demonstrated similar degrees of weight gain throughout the study period. End-point μ-computed tomography at 2 m post-op revealed no evidence of contrast extravasation, fistulas, strictures, or diverticula in any of the implant groups. Ex vivo tissue bath studies demonstrated that reconstructed esophageal conduits supported by both SF and SIS scaffolds displayed contractile responses to carbachol, KCl and electrical field stimulation while isoproterenol produced tissue relaxation. Histological (Masson's trichrome and hematoxylin and eosin) and immunohistochemical (IHC) evaluations demonstrated both implant groups produced de novo formation of skeletal and smooth muscle bundles positive for contractile protein expression [fast myosin heavy chain (MY32) and α-smooth muscle actin (α-SMA)] within the graft site. However, SF matrices promoted a significant 4-fold increase in MY32+ skeletal muscle and a 2-fold gain in α-SMA+ smooth muscle in comparison to the SIS cohort as determined by histomorphometric analyses. A stratified squamous, keratinized epithelium expressing cytokeratin 5 and involucrin proteins was also present at 2 m post-op in all experimental groups. De novo innervation and vascularization were evident in all regenerated tissues indicated by the presence of synaptophysin (SYP38)+ boutons and vessels lined with CD31 expressing endothelial cells. In respect to SIS, the SF group supported a significant 4-fold increase in the density of SYP38+ boutons within the implant region. Evaluation of host tissue responses revealed that SIS matrices elicited chronic inflammatory reactions and severe fibrosis throughout the neotissues, in contrast to SF scaffolds. The results of this study demonstrate that bi-layer SF scaffolds represent promising biomaterials for onlay esophagoplasty, capable of producing superior regenerative outcomes in comparison to conventional SIS scaffolds. [ABSTRACT FROM AUTHOR]

Abstract: Adverse side-effects associated with enterocystoplasty for neurogenic bladder reconstruction have spawned the need for the development of alternative graft substitutes. Bi-layer silk fibroin (SF) scaffolds and small intestinal submucosa (SIS) matrices were investigated for their ability to support bladder tissue regeneration and function in a rat model of spinal cord injury (SCI). Bladder augmentation was performed with each scaffold configuration in SCI animals for 10 wk of implantation and compared to non-augmented control groups (normal and SCI alone). Animals subjected to SCI alone exhibited a 72% survival rate (13/18) while SCI rats receiving SIS and bi-layer SF scaffolds displayed respective survival rates of 83% (10/12) and 75% (9/12) over the course of the study period. Histological (Masson's trichrome analysis) and immunohistochemical (IHC) evaluations demonstrated both implant groups supported de novo formation of smooth muscle layers with contractile protein expression [α-smooth muscle actin (α-SMA) and SM22α] as well as maturation of multi-layer urothelia expressing cytokeratin (CK) and uroplakin 3A proteins. Histomorphometric analysis revealed bi-layer SF and SIS scaffolds respectively reconstituted 64% and 56% of the level of α-SMA+ smooth muscle bundles present in SCI-alone controls, while similar degrees of CK+ urothelium across all experimental groups were detected. Parallel evaluations showed similar degrees of vascular area and synaptophysin+ boutons in all regenerated tissues compared to SCI-alone controls. In addition, improvements in certain urodynamic parameters in SCI animals, such as decreased peak intravesical pressure, following implantation with both matrix configurations were also observed. The data presented in this study detail the ability of acellular SIS and bi-layer SF scaffolds to support formation of innervated, vascularized smooth muscle and urothelial tissues in a neurogenic bladder model. [Copyright &y& Elsevier]

Abstract: Blended polymers are used extensively in many critical medical conditions as components of permanently implanted devices. Hybrid protein polymers containing recombinant human tropoelastin and silk fibroin have favorable characteristics as implantable scaffolds in terms of mechanical and biological properties. A firefly luciferase transgenic mouse model was used to monitor real-time IL-1β production localized to the site of biomaterial implantation, to observe the acute immune response (up to 5 days) to these materials. Significantly reduced levels of IL-1β were observed in silk/tropoelastin implants compared to control silk only implants at 1, 2 and 3 days post-surgery. Subsequently, mice (n = 9) were euthanized at 10 days (10D) and 3 weeks (3W) post-surgery to assess inflammatory cell infiltration and collagen deposition, using histopathology and immunohistochemistry. Compared to control silk only implants, fewer total inflammatory cells were found in silk/tropoelastin (∼29% at 10D and ∼47% at 3W). Also fewer ingrowth cells (∼42% at 10D and ∼63% at 3W) were observed within the silk/tropoelastin implants compared to silk only. Lower IL-6 (∼52%) and MMP-2 (∼84%) (pro-inflammatory) were also detected for silk/tropoelastin at 10 days. After 3 weeks implantation, reduced neovascularization (vWF ∼43%), fewer proliferating cells (Ki67 ∼58% and PCNA ∼41%), macrophages (F4/80 ∼64%), lower IL-10 (∼47%) and MMP-9 (∼55%) were also observed in silk/tropoelastin materials compared to silk only. Together, these results suggest that incorporation of tropoelastin improves on the established biocompatibility of silk fibroin, uniquely measured here as a reduced foreign body inflammatory response. [Copyright &y& Elsevier]

Abstract: Acellular scaffolds derived from Bombyx mori silk fibroin were investigated for their ability to support functional tissue regeneration in a porcine model of augmentation cystoplasty. Two bi-layer matrix configurations were fabricated by solvent-casting/salt leaching either alone (Group 1) or in combination with silk film casting (Group 2) to yield porous foams buttressed by heterogeneous surface pore occlusions or homogenous silk films, respectively. Bladder augmentation was performed with each scaffold group (6 × 6 cm2) in juvenile Yorkshire swine for 3 m of implantation. Augmented animals exhibited high rates of survival (Group 1: 5/6, 83%; Group 2: 4/4, 100%) and voluntary voiding over the course of the study period. Urodynamic evaluations demonstrated mean increases in bladder capacity over pre-operative levels (Group 1: 277%; Group 2: 153%) which exceeded nonsurgical control gains (144%) encountered due to animal growth. Similarly, elevations in bladder compliance were substantially higher in augmented animals from baseline (Group 1: 357%; Group 2: 147%) in comparison to controls (41%). Gross tissue evaluations revealed that both matrix configurations supported extensive de novo tissue formation throughout the entire original implantation site which exhibited ultimate tensile strength similar to nonsurgical counterparts. Histological and immunohistochemical analyses showed that both implant groups promoted comparable extents of smooth muscle regeneration and contractile protein (α-smooth muscle actin and SM22α) expression within defect sites similar to controls. Parallel evaluations demonstrated the formation of a transitional, multi-layered urothelium with prominent cytokeratin, uroplakin, and p63 protein expression in both matrix groups. De novo innervation and vascularization processes were evident in all regenerated tissues indicated by synaptophysin-positive neuronal cells and vessels lined with CD31 expressing endothelial cells. Ex vivo organ bath studies demonstrated that regenerated tissues supported by both silk matrices displayed contractile responses to carbachol, α,β-methylene-ATP, KCl, and electrical field stimulation similar to controls. Our data detail the ability of acellular silk scaffolds to support regeneration of innervated, vascularized smooth muscle and urothelial tissues within 3 m with structural, mechanical, and functional properties comparable to native tissue in a porcine model of bladder repair. [Copyright &y& Elsevier]

Abstract: Due to its unique properties, silk fibroin was studied as a biodegradable polymer vehicle for sustained, local delivery of the anticonvulsant adenosine from encapsulated reservoirs. Silk is a biologically derived protein polymer that is biocompatible, mechanically strong and degrades to non-toxic products in vivo. To achieve local, sustained, controlled adenosine release from fully degradable implants, solid adenosine powder reservoirs were coated with silk fibroin. Material properties of the silk coating including thickness, crystallinity and morphology were investigated to assess the relationships between silk coating biomaterial features and adenosine release from silk encapsulated reservoirs. Reservoir coating thickness was varied through manipulation of the silk coating solution concentration and number of coatings applied. Release studies were also performed in proteinase type XIV to model the effects of degradation. Increasing the barrier to diffusion, either by increasing coating thickness or crystallinity was found to delay adenosine burst, decrease average daily release rate, and increase duration of release. In the case of encapsulated reservoirs coated with eight layers of 8% (w/v) silk, a linear release profile was observed and adenosine release was sustained for 14days. The ability to achieve nearly constant release for 2weeks for adenosine via control of the silk coating suggests these encapsulated reservoirs represent a novel system for delivering adenosine. We anticipate that this approach could also be extended to other implant needs and small-molecule drugs to treat a range of clinical needs. [Copyright &y& Elsevier]

Abstract: Purified native silk fibroin forms β-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of β-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K+ at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca2+. The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5–2h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol–gel transitions, such as for cell encapsulation. [Copyright &y& Elsevier]