Rapid Volumetric Bioprinting of Decellularized Extracellular Matrix Bioinks.

Decellularized extracellular matrix (dECM)-based hydrogels are widely applied to additive biomanufacturing strategies for relevant applications. The extracellular matrix components and growth factors of dECM play crucial roles in cell adhesion, growth, and differentiation. However, the generally poor mechanical properties and printability have remained as major limitations for dECM-based materials. In this study, heart-derived dECM (h-dECM) and meniscus-derived dECM (Ms-dECM) bioinks in their pristine, unmodified state supplemented with the photoinitiator system of tris(2,2-bipyridyl) dichlororuthenium(II) hexahydrate and sodium persulfate, demonstrate cytocompatibility with volumetric bioprinting processes. This recently developed bioprinting modality illuminates a dynamically evolving light pattern into a rotating volume of the bioink, and thus decouples the requirement of mechanical strengths of bioprinted hydrogel constructs with printability, allowing for the fabrication of sophisticated shapes and architectures with low-concentration dECM materials that set within tens of seconds. As exemplary applications, cardiac tissues are volumetrically bioprinted using the cardiomyocyte-laden h-dECM bioink showing favorable cell proliferation, expansion, spreading, biomarker expressions, and synchronized contractions; whereas the volumetrically bioprinted Ms-dECM meniscus structures embedded with human mesenchymal stem cells present appropriate chondrogenic differentiation outcomes. This study supplies expanded bioink libraries for volumetric bioprinting and broadens utilities of dECM toward tissue engineering and regenerative medicine.

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.

Synthesis and characterization of silk-poly(guluronate) hybrid polymers for the fabrication of dual crosslinked, mechanically dynamic hydrogels.

The rapid ionic crosslinking of alginate has been actively studied for biomedical applications including hydrogel scaffolds for tissue engineering, injectable gels, and 3D bioprinting. However, the poor structural stability of ionic crosslinks under physiological conditions limits the widespread applications of these hydrogels. Moreover, the lack of cell adhesion to the material combined with the inability of proteases to degrade alginate further restrict utility as hydrogel scaffolds. Blends of alginate with silk fibroin have been proposed for improved structural and mechanical properties, but potential phase separation between the hydrophobic protein and the hydrophilic polysaccharide remains an issue. In this study, we demonstrated the synthesis of a hybrid biopolymer composed of a silk backbone with side chains of poly(guluronate) isolated from alginate to introduce rapid ionic crosslinking on enzymatically crosslinked silk-based hydrogels for on-demand and reversible stiffening and softening properties. Dual crosslinked macro- and microgels of silk fibroin-poly(guluronate) (SF-PG) hybrid polymers displayed dynamic morphology with reversible shrinking and swelling behavior. SF-PG hydrogel discs demonstrated dynamic mechanics with compressive moduli ranging from less than 5 kPa to over 80 kPa and underwent proteolytic degradation unlike covalently crosslinked alginate controls. SF-PG gels supplemented with gelatin substituted with tyramine or both tyramine and PG also supported the attachment and survival of murine fibroblasts, suggesting potential uses of these new hydrogels in mammalian cell culture to investigate cellular responses to dynamic mechanics or modeling of diseases defined by matrix mechanics, such as fibrosis and cancer.

Mucosa-Mimetic Materials for the Study of Intestinal Homeostasis and Disease.

Mucus is a viscoelastic hydrogel that lines and protects the epithelial surfaces of the body that houses commensal microbiota and functions in host defense against pathogen invasion. As a first-line physical and biochemical barrier, intestinal mucus is involved in immune surveillance and spatial organization of the microbiome, while dysfunction of the gut mucus barrier is implicated in several diseases. Mucus can be collected from a variety of mammalian sources for study, however, established methods are challenging in terms of scale and efficiency, as well as with regard to rheological similarity to native human mucus. Therefore, there is a need for mucus-mimetic hydrogels that more accurately reflect the physical and chemical profile of the in vivo human epithelial environment to enable the investigation of the role of mucus in human disease and interactions with the intestinal microbiome. This review will evaluate the material properties of synthetic mucus mimics to date designed to address the above need, with a focus toward an improved understanding of the biochemical and immunological functions of these biopolymers related to utility for research and therapeutic applications.

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.

Boronic Acid-Tethered Silk Fibroin for pH-Dependent Mucoadhesion.

Mucus lines all surfaces of the human body not covered by skin and provides lubrication, hydration, and protection. The properties of mucus are influenced by changes in pH that may occur due to physiological conditions and pathological circumstances. Reinforcing the mucus barrier with biopolymers that can adhere to mucus in different conditions can be a useful strategy for protecting the underlying mucosae from damage. In this work, regenerated silk fibroin (silk) was chemically modified with phenyl boronic acid to form reversible covalent complexes with the 1,2- or 1,3-diols. The silk modified with boronic acid pendant groups has an increased affinity for mucins, whose carbohydrate component is rich in diols. These results offer new applications of silk in mucoadhesion, and the ability to bind diols to the silk lays the foundation for the development of silk-based sugar-sensing platforms.

Volumetric additive manufacturing of pristine silk-based (bio)inks.

Volumetric additive manufacturing (VAM) enables fast photopolymerization of three-dimensional constructs by illuminating dynamically evolving light patterns in the entire build volume. However, the lack of bioinks suitable for VAM is a critical limitation. This study reports rapid volumetric (bio)printing of pristine, unmodified silk-based (silk sericin (SS) and silk fibroin (SF)) (bio)inks to form sophisticated shapes and architectures. Of interest, combined with post-fabrication processing, the (bio)printed SS constructs reveal properties including reversible as well as repeated shrinkage and expansion, or shape-memory; whereas the (bio)printed SF constructs exhibit tunable mechanical performances ranging from a few hundred Pa to hundreds of MPa. Both types of silk-based (bio)inks are cytocompatible. This work supplies expanded bioink libraries for VAM and provides a path forward for rapid volumetric manufacturing of silk constructs, towards broadened biomedical applications.

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.

Towards Non-stick Silk: Tuning the Hydrophobicity of Silk Fibroin Protein.

Silk fibroin protein is a biomaterial with excellent biocompatibility and low immunogenicity. These properties have catapulted the material as a leader for extensive use in stents, catheters, and wound dressings. Modulation of hydrophobicity of silk fibroin protein to further expand the scope and utility however has been elusive. We report that installing perfluorocarbon chains on the surface of silk fibroin transforms this water-soluble protein into a remarkably hydrophobic polymer that can be solvent-cast. A clear relationship emerged between fluorine content of the modified silk and film hydrophobicity. Water contact angles of the most decorated silk fibroin protein exceeded that of Teflon®. We further show that water uptake in prefabricated silk bars is dramatically reduced, extending their lifetimes, and maintaining mechanical integrity. These results highlight the power of chemistry under moderate conditions to install unnatural groups onto the silk fibroin surface and will enable further exploration into applications of this versatile biomaterial.

Cytoprotection of Human Progenitor and Stem Cells through Encapsulation in Alginate Templated, Dual Crosslinked Silk and Silk-Gelatin Composite Hydrogel Microbeads.

Susceptibility of mammalian cells against harsh processing conditions limit their use in cell transplantation and tissue engineering applications. Besides modulation of the cell microenvironment, encapsulation of mammalian cells within hydrogel microbeads attract attention for cytoprotection through physical isolation of the encapsulated cells. The hydrogel formulations used for cell microencapsulation are largely dominated by ionically crosslinked alginate (Alg), which suffer from low structural stability under physiological culture conditions and poor cell-matrix interactions. Here the fabrication of Alg templated silk and silk/gelatin composite hydrogel microspheres with permanent or on-demand cleavable enzymatic crosslinks using simple and cost-effective centrifugation-based droplet processing are demonstrated. The composite microbeads display structural stability under ion exchange conditions with improved mechanical properties compared to ionically crosslinked Alg microspheres. Human mesenchymal stem and neural progenitor cells are successfully encapsulated in the composite beads and protected against environmental factors, including exposure to polycations, extracellular acidosis, apoptotic cytokines, ultraviolet (UV) irradiation, anoikis, immune recognition, and particularly mechanical stress. The microbeads preserve viability, growth, and differentiation of encapsulated stem and progenitor cells after extrusion in viscous polyethylene oxide solution through a 27-gauge fine needle, suggesting potential applications in injection-based delivery and three-dimensional bioprinting of mammalian cells with higher success rates.

Molecularly cleavable bioinks facilitate high-performance digital light processing-based bioprinting of functional volumetric soft tissues.

Digital light processing bioprinting favors biofabrication of tissues with improved structural complexity. However, soft-tissue fabrication with this method remains a challenge to balance the physical performances of the bioinks for high-fidelity bioprinting and suitable microenvironments for the encapsulated cells to thrive. Here, we propose a molecular cleavage approach, where hyaluronic acid methacrylate (HAMA) is mixed with gelatin methacryloyl to achieve high-performance bioprinting, followed by selectively enzymatic digestion of HAMA, resulting in tissue-matching mechanical properties without losing the structural complexity and fidelity. Our method allows cellular morphological and functional improvements across multiple bioprinted tissue types featuring a wide range of mechanical stiffness, from the muscles to the brain, the softest organ of the human body. This platform endows us to biofabricate mechanically precisely tunable constructs to meet the biological function requirements of target tissues, potentially paving the way for broad applications in tissue and tissue model engineering.

Cell-controlled dynamic surfaces for skeletal stem cell growth and differentiation.

Skeletal stem cells (SSCs, or mesenchymal stromal cells typically referred to as mesenchymal stem cells from the bone marrow) are a dynamic progenitor population that can enter quiescence, self-renew or differentiate depending on regenerative demand and cues from their niche environment. However, ex vivo, in culture, they are grown typically on hard polystyrene surfaces, and this leads to rapid loss of the SSC phenotype. While materials are being developed that can control SSC growth and differentiation, very few examples of dynamic interfaces that reflect the plastic nature of the stem cells have, to date, been developed. Achieving such interfaces is challenging because of competing needs: growing SSCs require lower cell adhesion and intracellular tension while differentiation to, for example, bone-forming osteoblasts requires increased adhesion and intracellular tension. We previously reported a dynamic interface where the cell adhesion tripeptide arginine-glycine-aspartic acid (RGD) was presented to the cells upon activation by user-added elastase that cleaved a bulky blocking group hiding RGD from the cells. This allowed for a growth phase while the blocking group was in place and the cells could only form smaller adhesions, followed by an osteoblast differentiation phase that was induced after elastase was added which triggered exposure of RGD and subsequent cell adhesion and contraction. Here, we aimed to develop an autonomous system where the surface is activated according to the need of the cell by using matrix metalloprotease (MMP) cleavable peptide sequences to remove the blocking group with the hypothesis that the SSCs would produce higher levels of MMP as the cells reached confluence. The current studies demonstrate that SSCs produce active MMP-2 that can cleave functional groups on a surface. We also demonstrate that SSCs can grow on the uncleaved surface and, with time, produce osteogenic marker proteins on the MMP-responsive surface. These studies demonstrate the concept for cell-controlled surfaces that can modulate adhesion and phenotype with significant implications for stem cell phenotype modulation.

3D Printing of Monolithic Proteinaceous Cantilevers Using Regenerated Silk Fibroin.

Silk fibroin, regenerated from Bombyx mori, has shown considerable promise as a printable, aqueous-based ink using a bioinspired salt-bath system in our previous work. Here, we further developed and characterized silk fibroin inks that exhibit concentration-dependent fluorescence spectra at the molecular level. These insights supported extrusion-based 3D printing using concentrated silk fibroin solutions as printing inks. 3D monolithic proteinaceous structures with high aspect ratios were successfully printed using these approaches, including cantilevers only supported at one end. This work provides further insight and broadens the utility of 3D printing with silk fibroin inks for the microfabrication of proteinaceous structures.

Challenges in delivering therapeutic peptides and proteins: A silk-based solution.

Peptide- and protein-based therapeutics have drawn significant attention over the past few decades for the treatment of infectious diseases, genetic disorders, oncology, and many other clinical needs. Yet, protecting peptide- and protein-based drugs from degradation and denaturation during processing, storage and delivery remain significant challenges. In this review, we introduce the properties of peptide- and protein-based drugs and the challenges associated with their stability and delivery. Then, we discuss delivery strategies using synthetic polymers and their advantages and limitations. This is followed by a focus on silk protein-based materials for peptide/protein drug processing, storage, and delivery, as a path to overcome stability and delivery challenges with current systems.

Fiber-Based Biopolymer Processing as a Route toward Sustainability.

Some of the most abundant biomass on earth is sequestered in fibrous biopolymers like cellulose, chitin, and silk. These types of natural materials offer unique and striking mechanical and functional features that have driven strong interest in their utility for a range of applications, while also matching environmental sustainability needs. However, these material systems are challenging to process in cost-competitive ways to compete with synthetic plastics due to the limited options for thermal processing. This results in the dominance of solution-based processing for fibrous biopolymers, which presents challenges for scaling, cost, and consistency in outcomes. However, new opportunities to utilize thermal processing with these types of biopolymers, as well as fibrillation approaches, can drive renewed opportunities to bridge this gap between synthetic plastic processing and fibrous biopolymers, while also holding sustainability goals as critical to long-term successful outcomes.

Sugar Functionalization of Silks with Pathway-Controlled Substitution and Properties.

Silk biomaterials are important for applications in biomedical fields due to their outstanding mechanical properties, biocompatibility, and tunable biodegradation. Chemical functionalization of silk by various chemistries can be leveraged to enhance and tune these features and enable the expansion of silk-based biomaterials into additional fields. Sugars are particularly relevant for intracellular communication, signal transduction events, as well as in hydrated extracellular matrices such as in cartilage, vitreous, and brain tissues. Multiple reaction pathways are demonstrated (carboxylation of serines followed by carbodiimide coupling with glucosamine, carboxylation of tyrosines followed by carbodiimide coupling with glucosamine; direct carbodiimide coupling of the inherent carboxylic acids of silk (aspartic and glutamic acid) with glucosamine) for the covalent conjugation of glucosamine onto silk with characterization by proton nuclear magnetic resonance (1 H-NMR), liquid chromatography tandem mass spectroscopy (LC-MS), water contact angle (WCA), and Fourier transform infrared (FTIR) spectroscopy. The results indicate that different pathways substitute different amounts of glucosamine onto silk chains, with control over resulting material properties, including hydrophobicity/hydrophilicity and biological responses. The aqueous processability of these conjugates into functional material formats (films, sponges) is assessed. These new classes of bio-inspired materials can lead to multifunctional biomaterials for potential applications in different fields of biomedical engineering.

Silk Hydrogels with Controllable Formation of Dityrosine, 3,4-Dihydroxyphenylalanine, and 3,4-Dihydroxyphenylalanine-Fe3+ Complexes through Chitosan Particle-Assisted Fenton Reactions.

The oxidation of tyrosine residues of silk fibroin involves the generation of dityrosine and 3,4-dihydroxyphenylalanine (DOPA). However, it remains a challenge to selectively control the reaction pathway to produce dityrosine or DOPA in a selective fashion. Here, silk hydrogels with controllable formation of not only dityrosine and DOPA but also DOPA-Fe3+ complexes within the cross-linked networks were developed. The use of chitosan particles in the Fenton reaction allowed the interaction of Fe3+ ions with silk fibroin to be limited through the adsorption of Fe3+ ions onto chitosan particles by manipulating contact time between the reaction medium and chitosan particles. This led to significant suppression of the premature formation of ß-sheet structures that cause steric hindrance to the collisions between tyrosyl radicals and thus enabled higher selectivity toward the formation of dityrosine than DOPA. Remarkably, the addition of ethylenediaminetetraacetic acid (EDTA) to the chitosan particle-assisted Fenton reactions resulted in hydrogels that significantly favored the formation of DOPA over dityrosine due to the increase in the hydroxylation of phenol in the presence of EDTA. Despite the existence of Fe3+-EDTA complexes, Raman spectra indicated the DOPA-Fe3+ complexation in the hydrogels. Mechanistically, the hydrogel networks with small-sized and uniformly distributed ß-sheet structures as well as the abundance of DOPA appear to make non-EDTA-chelated Fe3+ ions more accessible to complexation with DOPA. These findings have important implications for understanding the oxidation of tyrosine residues of silk fibroin by metal-catalyzed oxidation systems with potential benefits for future studies on silk protein-based hydrogels capable of generating intrinsic adhesive features as well as for exploring dual-cross-linked silk hydrogels constructed by chemical cross-linking and metal-coordinate complexation.

Photo-Crosslinked Silk Fibroin for 3D Printing.

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.

Silk degumming time controls horseradish peroxidase-catalyzed hydrogel properties.

Hydrogels provide promising applications in tissue engineering and regenerative medicine, with silk fibroin (SF) offering biocompatibility, biodegradability and tunable mechanical properties. The molecular weight (MW) distribution of SF chains varies from ∼80 to 400 kDa depending on the extraction and purification process utilized to prepare the protein polymer. Here, we report a fundamental study on the effect of different silk degumming (extraction) time (DT) on biomaterial properties of enzymatically crosslinked hydrogels, including secondary structure, mechanical stiffness, in vitro degradation, swelling/contraction, optical transparency and cell behaviour. The results indicate that DT plays a crucial role in determining material properties of the hydrogel; decrease in DT increases ß-sheet (crystal) formation and mechanical stiffness while decreasing degradation rate and optical transparency. The findings on the relationships between properties of silk hydrogels and DT should facilitate the more rational design of silk-based hydrogel biomaterials to match properties needed for diverse purpose in biomedical engineering.