Instantaneous Formation of Silk Protein Aerosols and Fibers with a Portable Spray Device Under Ambient Conditions.

A variety of artificial silk spinning approaches have been 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, we report a new strategy to fabricate silk protein-based aerosols and silk fibers instantaneously (< 1 s) in situ using a simple, portable, 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 ß-sheet right before spraying by mixing silk and polyethylene glycol (PEG) solutions in the spray device, allowing aerosols and silk fibers to be sprayed in situ, 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, i.e., 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.

Engineered porosity for tissue-integrating, bioresorbable lifetime-based phosphorescent oxygen sensors.

Versatile silk protein-based material formats were studied to demonstrate bioresorbable, implantable optical oxygen sensors that can integrate with the surrounding tissues. The ability to continuously monitor tissue oxygenation in vivo is desired for a range of medical applications. Silk was chosen as the matrix material due to its excellent biocompatibility, its unique chemistry that facilitates interactions with chromophores, and the potential to tune degradation time without altering chemical composition. A phosphorescent Pd (II) benzoporphyrin chromophore was incorporated to impart oxygen sensitivity. Organic solvent-based processing methods using 1,1,1,3,3,3-hexafluoro-2-propanol were used to fabricate: 1) silk-chromophore films with varied thickness and 2) silk-chromophore sponges with interconnected porosity. All compositions were biocompatible and exhibited photophysical properties with oxygen sensitivities (i.e., Stern-Volmer quenching rate constants of 2.7-3.2 × 104 M-1) useful for monitoring physiological tissue oxygen levels and for detecting deviations from normal behavior (e.g., hyperoxia). The potential to tune degradation time without significantly impacting photophysical properties was successfully demonstrated. Furthermore, the ability to consistently monitor tissue oxygenation in vivo was established via a multi-week rodent study. Histological assessments indicated successful tissue integration for the sponges, and this material format responded more quickly to various oxygen challenges than the film samples.

Multifunctional silk vinyl sulfone-based hydrogel scaffolds for dynamic material-cell interactions.

Biochemical and mechanical interactions between cells and the surrounding extracellular matrix influence cell behavior and fate. Mimicking these features in vitro has prompted the design and development of biomaterials, with continuing efforts to improve tailorable systems that also incorporate dynamic chemical functionalities. The majority of these chemistries have been incorporated into synthetic biomaterials, here we focus on modifications of silk protein with dynamic features achieved via enzymatic, "click", and photo-chemistries. The one-pot synthesis of vinyl sulfone modified silk (SilkVS) can be tuned to manipulate the degree of functionalization. The resultant modified protein-based material undergoes three different gelation mechanisms, enzymatic, "click", and light-induced, to generate hydrogels for in vitro cell culture. Further, the versatility of this chemical functionality is exploited to mimic cell-ECM interactions via the incorporation of bioactive peptides and proteins or by altering the mechanical properties of the material to guide cell behavior. SilkVS is well-suited for use in in vitro culture, providing a natural protein with both tunable biochemistry and mechanics.

Silk chemistry and biomedical material designs.

Silk fibroin has applications in different medical fields such as tissue engineering, regenerative medicine, drug delivery and medical devices. Advances in silk chemistry and biomaterial designs have yielded exciting tools for generating new silk-based materials and technologies. Selective chemistries can enhance or tune the features of silk, such as mechanics, biodegradability, processability and biological interactions, to address challenges in medically relevant materials (hydrogels, films, sponges and fibres). This Review details the design and utility of silk biomaterials for different applications, with particular focus on chemistry. This Review consists of three segments: silk protein fundamentals, silk chemistries and functionalization mechanisms. This is followed by a description of different crosslinking chemistries facilitating network formation, including the formation of composite biomaterials. Utility in the fields of tissue engineering, drug delivery, 3D printing, cell coatings, microfluidics and biosensors are highlighted. Looking to the future, we discuss silk biomaterial design strategies to continue to improve medical outcomes.

Sudan Black B Pretreatment to Suppress Autofluorescence in Silk Fibroin Scaffolds.

Natural polymers are extensively utilized as scaffold materials in tissue engineering and 3D disease modeling due to their general features of cytocompatibility, biodegradability, and ability to mimic the architecture and mechanical properties of the native tissue. A major limitation of many polymeric scaffolds is their autofluorescence under common imaging methods. This autofluorescence, a particular challenge with silk fibroin materials, can interfere with the visualization of fluorescently labeled cells and proteins grown on or in these scaffolds, limiting the assessment of outcomes. Here, Sudan Black B (SBB) was successfully used prefixation prior to cell seeding, in various silk matrices and 3D model systems to quench silk autofluorescence for live cell imaging. SBB was also trialed postfixation in silk hydrogels. We validated that multiple silk scaffolds pretreated with SBB (hexafluoro-2-propanol-silk scaffolds, salt-leached sponges, gel-spun catheters, and sponge-gel composite scaffolds) cultured with fibroblasts, adipose tissue, neural cells, and myoblasts demonstrated improved image resolution when compared to the nonpretreated scaffolds, while also maintaining normal cell behavior (attachment, growth, proliferation, differentiation). SBB pretreatment of silk scaffolds is an option for scaffold systems that require autofluorescence suppression.

Horseradish Peroxidase Catalyzed Silk-Prefoldin Composite Hydrogel Networks.

Protein-based hydrogel biomaterials provide a platform for different biological applications, including the encapsulation and stabilization of different biomolecules. These hydrogel properties can be modulated by controlling the design parameters to match specific needs; thus, multicomponent hydrogels have distinct advantages over single-component hydrogels due to their enhanced versatility. Here, silk fibroin and γ-prefoldin chaperone protein based composite hydrogels were prepared and studied. Different ratios of the proteins were chosen, and the hydrogels were prepared by enzyme-assisted cross-linking. The secondary structure of the two proteins, dityrosine bond formation, and mechanical properties were assessed. The results obtained can be used as a platform for the rational design of composite thermostable hydrogel biomaterials to facilitate protection (due to hydrogel mechanics) and retention of bioactivity (e.g., of enzymes and other biomolecules) due to chaperone-like properties of γ-prefoldin.

Emerging Trajectories for Next Generation Tissue Engineers.

The field of tissue engineering has evolved from its early days of engineering tissue substitutes to current efforts at building human tissues for regenerative medicine and mechanistic studies of tissue disease, injury, and regeneration. Advances in bioengineering, material science, and stem cell biology have enabled major developments in the field. In this perspective, we reflect on the September 2021 virtual Next Generation Tissue Engineering symposium and trainee workshop, as well as our projections for the field over the next 15 years.