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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 × 10 4  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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Aging-associated neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases remain poorly understood and no disease-modifying treatments exist despite decades of investigation. Predominant in vitro (e.g., 2D cell culture, organoids) and in vivo (e.g., mouse) models of these diseases are insufficient mimics of human brain tissue structure and function and of human neurodegenerative pathobiology, and have thus contributed to this collective translational failure. This has been a longstanding challenge in the field, and new strategies are required to address both fundamental and translational needs. Bioengineered tissue culture models constitute a class of promising alternatives, as they can overcome the low cell density, poor nutrient exchange, and long term culturability limitations of existing in vitro models. Further, they can reconstruct the structural, mechanical, and biochemical cues of native brain tissue, providing a better mimic of human brain tissues for in vitro pathobiological investigation and drug development. We discuss bioengineering techniques for the generation of these neurodegenerative tissue models, including biomaterials-, organoid-, and microfluidics-based approaches, and design considerations for their construction. To aid the development of the next generation of functional neurodegenerative disease models, we discuss approaches to incorporate greater cellular diversity and simulate aging processes within bioengineered brain tissues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The development of cost-effective serum-free media is essential for the economic viability of cultured meat. A key challenge facing this goal is the high-cost of recombinant albumin which is necessary in many serum-free media formulations, including a recently developed serum-free medium for bovine satellite cell (BSC) culture termed Beefy-9. Here we alter Beefy-9 by replacing recombinant albumin with rapeseed protein isolate (RPI), a bulk-protein solution obtained from agricultural waste through alkali extraction (pH 12.5), isoelectric protein precipitation (pH 4.5), dissolution of physiologically soluble proteins (pH 7.2), and concentration of proteins through 3 kDa ultrafiltration. This new medium, termed Beefy-R, was then used to culture BSCs over four passages, during which cells grew with an average doubling time of 26.6 h, showing improved growth compared with Beefy-9. In Beefy-R, BSCs maintained cell phenotype and myogenicity. Together, these results offer an effective, low-cost, and sustainable alternative to albumin for serum-free culture of muscle stem cells, thereby addressing a key hurdle facing cultured meat production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Collagen presents a well-organized hierarchical multilevel structure. Microfibers, fibers, and fiber bundles are the aggregates of natural collagen; which achieve an ideal balance of mechanical strength and toughness at the mesoscopic scale for biological tissue. These mesostructured aggregates of collagen isolated from biological tissues retain these inherent organizational features to enable their use as building blocks for constructing new collagen materials with ideal mechanical performance, thermal and dimensional stability. This strategy is distinct from the more common bottom-up or molecular-level design and assembly approach to generating collagen materials. The present review introduces the hierarchical structure of biological collagen with a focus on mesostructural features. Isolation strategies for these collagen aggregates (CAs) are summarized. Recent progress in the use of these mesostructural components for the construction of new collagen materials with emerging applications is reviewed, including in catalysis, environmental applications, biomedicine, food packaging, electrical energy storage, and flexible sensors. Finally, challenges and prospects are assessed for controllable production of CAs as well as material designs., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled “Collagen Processing with Mesoscale Aggregates as Templates and Building Blocks”., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Recombinant spider silk materials with antimicrobial peptides are a promising new class of drug-free antimicrobial materials capable of preventing surgical site infections (SSI), but their potential to impede infections is unclear. Herein, we aimed to unravel the biological and inflammatory potential of bioengineered spider silk materials to prevent SSI using an infection animal model. Silk-like fibers made of silk fibroin and spider silk proteins with antimicrobial peptides (6mer-HNP1) held improved stiffness (2.9 GPa) and had a slow biodegradation profile while inhibiting bacterial adherence in vitro by 5-log and 6-log reduction on Methicillin-Resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. In vivo studies showed that fibers with 6mer-HNP1 elicited a short-term low to mild local inflammatory response, similar to implanted commercial sutures. In the presence of a bacterial infection, the mediators related to infection and inflammation were downregulated suggesting that the fibers maintained a low but active response to bacterial infection. Thus, the presence of 6mer-HNP1 helped the host maintain an active response to bacterial infection, impairing the development of an acute infection. Our findings further support the use of bioengineered spider silk proteins to develop drug-free antimicrobial sutures capable to impair SSI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Studies of underlying neurodegenerative processes in Parkinson's Disease (PD) have traditionally utilized cell cultures grown on two-dimensional (2D) surfaces. Biomimetic three-dimensional (3D) cell culture platforms have been developed to better emulate features of the brain's natural microenvironment. We here use our bioengineered brain-like tissue model, composed of a silk-hydrogel composite, to study the 3D microenvironment's contributions on the development and performance of dopaminergic-like neurons (DLNs). Compared with 2D culture, SH-SY5Y cells differentiated in 3D microenvironments were enriched for DLNs concomitant with a reduction in proliferative capacity during the neurodevelopmental process. Additionally, the 3D DLN cultures were more sensitive to oxidative stresses elicited by the PD-related neurotoxin 1-methyl-4-phenylpyridinium (MPP). MPP induced transcriptomic profile changes specific to 3D-differentiated DLN cultures, replicating the dysfunction of neuronal signaling pathways and mitochondrial dynamics implicated in PD. Overall, this physiologically-relevant 3D platform resembles a useful tool for studying dopamine neuron biology and interrogating molecular mechanisms underlying neurodegeneration in PD., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: David Kaplan reports financial support was provided by National Institutes of Health., (Copyright © 2022. Published by Elsevier Ltd.)

Biomaterial scaffolds are critical components in cultivated meat production for enabling cell adhesion, proliferation, differentiation and orientation. Currently, there is limited information on the fabrication of edible/biodegradable scaffolds for cultivated meat applications. In the present work, several abundant, naturally derived biomaterials (gelatin, soy, glutenin, zein, cellulose, alginate, konjac, chitosan) were fabricated into films without toxic cross-linking or stabilizing agents. These films were investigated for support of the adhesion, proliferation and differentiation of murine and bovine myoblasts. These biomaterials supported cell viability, and the protein-based films showed better cell adhesion than the polysaccharide-based films. Surface patterns induced cell alignment and guided myoblast differentiation and organization on the glutenin and zein films. The mechanical properties of the protein films were also assessed and suggested that a range of properties can be achieved to meet food-related goals. Overall, based on adherence, proliferation, differentiation, mechanics, and material availability, protein-based films, particularly glutenin and zein, showed the most promise for cultivated meat applications. Ultimately, this work presents a comparison of suitable biomaterials for cultivated meat applications and suggests future efforts to optimize scaffolds for efficacy and cost., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

3D printing has experienced swift growth for biological applications in the field of regenerative medicine and tissue engineering. Essential features of bioprinting include determining the appropriate bioink, printing speed mechanics, and print resolution while also maintaining cytocompatibility. However, the scarcity of bioinks that provide printing and print properties and cell support remains a limitation. Silk Fibroin (SF) displays exceptional features and versatility for inks and shows the potential to print complex structures with tunable mechanical properties, degradation rates, and cytocompatibility. Here we summarize recent advances and needs with the use of SF protein from Bombyx mori silkworm as a bioink, including crosslinking methods for extrusion bioprinting using SF and the maintenance of cell viability during and post bioprinting. Additionally, we discuss how encapsulated cells within these SF-based 3D bioprinted constructs are differentiated into various lineages such as skin, cartilage, and bone to expedite tissue regeneration. We then shift the focus towards SF-based 3D printing applications, including magnetically decorated hydrogels, in situ bioprinting, and a next-generation 4D bioprinting approach. Future perspectives on improvements in printing strategies and the use of multicomponent bioinks to improve print fidelity are also discussed., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Alzheimer's Disease (AD) is a neurodegenerative disorder that can cause life-altering and debilitating cognitive decline. AD's etiology is poorly understood, and no disease-modifying therapeutics exist. Here, we describe the use of 2D and 3D tissue culture models of herpesvirus-induced AD, which recapitulate hallmark disease features of plaque formation, gliosis, neuroinflammation, and impaired neuronal signaling, to screen a panel of 21 medications, supplements, and nutraceuticals with purported neuroprotective benefits. This screen identified green tea catechins and resveratrol as having strong anti-plaque properties, functional neuroprotective benefits, and minimal neurotoxicity, providing support for their further investigation as AD preventives and therapies. Two other candidates, citicoline and metformin, reduced plaque formation and were minimally toxic, but did not protect against virus-induced impairments in neuronal signaling. This study establishes a simple platform for rapidly screening and characterizing AD compounds of interest in 2D and 3D human cortical tissue models representing physiologically relevant disease features., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Scaffolds suitable for use in food products are crucial components for the production of cultured meat. Here, wheat glutenin, an inexpensive and abundant plant-based protein, was used to develop 3D porous scaffolds for cultured meat applications. A physical cross-linking method based on water annealing was developed for the fabrication of porous glutenin sponges and fibrous aligned scaffolds. The pore sizes ranged from 50 to 250 μm, with compressive modulus ranges from 0.5 to 1.9 kPa, depending on the percentage of glutenin (2%-5%) used in the process. The sponges were stable in PBS with refrigeration for at least six months after water annealing. The glutenin scaffolds supported the proliferation and differentiation of C2C12 mouse skeletal myoblasts and bovine satellite cells (BSCs) without the need to add specific cell adhesive proteins or other coatings. The low cost and food safe production process avoided the use of toxic cross-linkers and animal-derived extracellular matrix (ECM) coatings, suggesting that this as approach is a promising system for scaffolds useful in cultivated meat applications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Our goal was to generate functionalized 3D-printed scaffolds for bone regeneration using silk-hydroxyapatite bone cements and osteoinductive, proangiogenic and neurotrophic growth factors or morphogens for accelerated bone formation. 3D printing was utilized to generate macroporous scaffolds with controlled geometries and architectures that promote osseointegration. We build on the knowledge that the osteoinductive factor Bone Morphogenetic Protein-2 (BMP2) can also positively impact vascularization, Vascular Endothelial Growth Factor (VEGF) can impact osteoblastic differentiation, and that Neural Growth Factor (NGF)-mediated signaling can influence bone regeneration. We assessed functions on the 3D printed construct via the osteogenic differentiation of human mesenchymal stem cells; migration and proliferation of human umbilical vein endothelial cells; and proliferation of human induced neural stem cells. The scaffolds provided mechanical properties suitable for bone and the materials were cytocompatible, osteoconductive and maintained the activity of the morphogens and cytokines. Synergistic outcomes between BMP-2, VEGF and NGF in terms of osteoblastic differentiation in vitro were identified, based on the upregulation of genes associated with osteoblastic differentiation (Runt-related transcription factor-2, Osteopontin, Bone Sialoprotein). Additional studies will be required to assess these scaffold designs in vivo. These results are expected to have a strong impact in bone regeneration in dental, oral and maxillofacial surgery., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bioengineered neural tissues help advance our understanding of neurodevelopment, regeneration, and neural disease; however, it remains unclear whether they can replicate higher-order functions including cognition. Building upon technical achievements in the fields of biomaterials, tissue engineering, and cell biology, investigators have generated an assortment of artificial brain structures and cocultured circuits. Though they have displayed basic electrochemical signaling, their capacities to generate minimal patterns of information processing suggestive of high-order cognitive analogues have not yet been explored. Here, we review the current state of neural tissue engineering and consider the possibility of a study of cognition in vitro. We adopt a practical definition of minimal cognition, anticipate problems of measurement, and discuss solutions toward a study of cognition in a dish., Competing Interests: Declaration of Interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Traditional bone fixation devices made from inert metal alloys provide structural strength for bone repair but are limited in their ability to actively promote bone healing. Although several naturally derived bioactive materials have been developed to promote ossification in bone defects, it is difficult to translate small-scale benchtop fabrication of these materials to high-output manufacturing. Standard industrial molding processes, such as injection and compression molding, have typically been limited to use with synthetic polymers since most biopolymers cannot withstand the harsh processing conditions involved in these techniques. Here we demonstrate injection and compression molding of a bioceramic composite comprised of hydroxyapatite (HA) and silk fibroin (SF) from Bombyx mori silkworm cocoons. Both the molding behavior of the HA-SF slurry and final scaffold mechanics can be controlled by modulating SF protein molecular weight, SF content, and powder-to-liquid ratio. HA-SF composites with up to 20 weight percent SF were successfully molded into stable three-dimensional structures using high pressure molding techniques. The unique durability of silk fibroin enables application of common molding techniques to fabricate composite silk-ceramic scaffolds. This work demonstrates the potential to move bone tissue engineering one step closer to large-scale manufacturing of natural protein-based resorbable bone grafts and fixation devices., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Patients with Alport syndrome (AS) exhibit blood and elevated protein levels in their urine, inflamed kidneys, and many other abnormalities. AS is attributed to mutations in type IV collagen genes, particularly glycine missense mutations in the collagenous domain of COL4A5 that disrupt common structural motifs in collagen from the repeat (Gly-Xaa-Yaa) n amino acid sequence. To characterize and elucidate the molecular mechanisms underlying how AS-related mutations perturb the structure and function of type IV collagen, experimental studies and molecular simulations were integrated to investigate the structure, stability, protease sensitivity, and integrin binding affinity of collagen-like proteins containing amino acid sequences from the α5(IV) chain and AS-related Gly missense mutations. We show adverse effects where (i) three AS-related Gly missense mutations significantly reduced the structural stability of the collagen in terms of decreased melting temperatures and calorimetric enthalpies, in conjunction with a collective drop in the external work needed to unfold the peptides containing mutation sequences; (ii) due to local unwinding around the sites of mutations, these triple helical peptides were also degraded more rapidly by trypsin and chymotrypsin, as these enzymes could access the collagenous triple helix more easily and increase the number of contacts; (iii) the mutations further abolished the ability of the recombinant collagens to bind to integrins and greatly reduced the binding affinities between collagen and integrins, thus preventing cells from adhering to these mutants. Our unified experimental and computational approach provided underlying insights needed to guide potential therapies for AS that ameliorate the adverse effects from AS disease onset and progression., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Multiple ophthalmic pathologies, such as retinal detachment and diabetic retinopathy, require the removal and replacement of the vitreous humor. Clinical tamponades such as silicone oil and fluorinated gases are utilized but limited due to complications and toxicity. Therefore, there is a need for biocompatible, stable, vitreous humor substitutes. In this study, enzymatically crosslinked silk-hyaluronic acid (HA) hydrogels formed using horseradish peroxidase and H 2 O 2 were characterized for use as vitreous humor substitutes. The composite network structure was characterized with dynamic light scattering. In addition, the rheological, optical, and swelling properties of hydrogels with varying silk to HA ratios and crosslinking densities controlled via H 2 O 2 were determined over time. Hydrogels had refractive indexes of 1.336 and were clear with 75-91% light transmission. Hydrogel shear storage modulus ranged between ~6 and 240 Pa where increased H 2 O 2 increased the modulus. After 1 month of aging, there were no changes in modulus for hydrogels with lower silk ratios, while those with higher silk ratios exhibited a significant increase in modulus. Decreasing H 2 O 2 concentration in the reactions led to increased hydrogel volume during swelling, with higher silk ratios returning to their original size after 15 days. Dynamic light scattering results show three diffusive modes, revealing the possible structures of the hydrogel composite and are consistent with the mechanical properties and swelling results. The normalized intraocular pressure of ex vivo porcine eyes after injecting hydrogels were comparable with those treated with silicone oil showing the potential clinical utility of the hydrogels as vitreous substitutes. The versatility of the silk-HA hydrogel system, the tunable swelling properties, and the stability of hydrogels with lower silk ratios show the benefit of utilizing silk-HA hydrogels as vitreous substitutes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

There is a large unmet need for off-the-shelf biomaterial options to supplant venous autografts in bypass and reconstructive surgical procedures. Existing graft alternatives formed from non-degradable synthetic polymers are not capable of maintaining long-term patency and are thus not indicated for <6 mm inner diameter bypass procedures. To fill this void, degradable silk-based biomaterials have been proposed that can maintain their mechanical properties (i.e. compliance) while facilitating slow but progressive biomaterial remodeling and host integration mediated by cellular colonization. The goal of the present study was to enhance the porosity of gel-spun silk tubes, to facilitate faster degradation rates and improve cellularity, and thus improve host integration over time in vivo, while maintaining requisite mechanical functions. Silk solutions with a range of molecular weight distributions and, in turn, viscosities were used to generate tubes of varying porosities. A decrease in solution concentration correlated with an increase in mean pore size and overall porosity through a density-dependent mechanism. Tubes were mechanically analyzed, and these properties were the basis of an analytical model used to correlate tube formulations to structural compliance, which were shown to be similar to the saphenous vein. Tubes were also tested for suture retention to ensure surgical utility despite increased porosity. Tubes were implanted in the abdominal aorta of Sprague-Dawley rats via an end-to-end anastomosis model. Tubes with higher porosities showed early improvements in cell colonization that progressively increased over time; conversely, the dense architecture of less porous grafts (20MB) inhibited cell ingrowth and resulted in minimal biomaterial degradation at the 6-month time point. None of the highly porous tubes (5 MB and 10MB) remained patent at 6 months, likely due remodeling inducing bulk mechanical failure or a compromised blood-material interface., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Silk fibroin (SF) was enzymatically crosslinked with tyramine-substituted silk fibroin (SF-TA) or gelatin (G-TA) to fabricate hybrid hydrogels with tunable gelation kinetics, mechanical properties and bioactivity. Horseradish peroxidase (HRP)/hydrogen peroxide (H 2 O 2 ) mediated crosslinking of SF in physiological buffers results in slow gelation and limited mechanical properties. Moreover, SF lacks cell attachment sequences, leading to poor cell-material interactions. These shortcomings can limit the uses of enzymatically crosslinked silk hydrogels in injectable tissue fillings, 3D bioprinting or cell microencapsulation, where rapid gelation and high bioactivity are desired. Here SF/SF-TA and SF/G-TA composite hydrogels were characterized for hydrogel properties and the influence of conjugated cyclic arginine-glycine-aspartic acid (RGD) peptide or G-TA content on bioactivity was explored. Both SF-TA and G-TA significantly increased gelation kinetics, improved mechanical properties and delayed enzymatic degradation in a concentration-dependent manner. β-Sheet formation and hydrogel stiffening were accelerated by SF-TA content but delayed by G-TA. Both cyclic RGD and G-TA significantly improved morphology and metabolic activity of human mesenchymal stem cells (hMSCs) cultured on or encapsulated in composite hydrogels. The hydrogel formulations introduced in this study provide improved control of gel formation and properties, along with biocompatible systems that can be utilized in tissue engineering and cell delivery applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bioengineering approaches grounded in immunology have the potential for the discovery and development of a successful HIV vaccine. The overarching goal is to engineer immunity through a fusion of immunology with bioengineering to create novel strategies for the design, development and delivery of vaccines based on the controlled modulation of the immune system. To foster these collaborations, the National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Biomedical Imaging and Bioengineering (NIBIB) brought together a group of experts (see Table 1) from these diverse fields for a workshop in September 2018 to: (1) engage the engineering, immunology, and HIV vaccinology communities to dialogue on the topic of an HIV vaccine and; (2) generate a framework of new and innovative research avenues to explore in HIV vaccinology between knowledge stakeholders and problem solvers., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

An in vitro model of intestinal epithelium with an immune component was bioengineered to mimic immunologic responses seen in inflammatory bowel disease. While intestinal immune phenomena can be modeled in transwells and 2D culture systems, 3D tissue models improve physiological relevance by providing a 3D substrate which enable migration of macrophages towards the epithelium. An intestinal epithelial layer comprised of non-transformed human colon organoid cells and a subepithelial layer laden with monocyte-derived macrophages was bioengineered to mimic native intestinal mucosa cell organization using spongy biomaterial scaffolds. Confluent monolayers with microvilli, a mucus layer, and infiltration of macrophages to the basal side of the epithelium were observed. Inflammation, induced by E. coli O111:B4 lipopolysaccharide and interferon γ resulted in morphological changes to the epithelium, resulting in ball-like structures, decreased epithelial coverage, and increased migration of macrophages to the epithelium. Analysis of cytokines present in the inflamed tissue model demonstrated significantly upregulated secretion of pro-inflammatory cytokines that are often associated with active inflammatory bowel disease, including CXCL10, IL-1β, IL-6, MCP-2, and MIP-1β. The macrophage layer enhanced epithelial and biochemical responses to inflammatory insult, and this new tissue system may be useful to study and develop potential therapies for inflammatory bowel disease., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Current commercially available human skin equivalents (HSEs) are used for relatively short term studies (∼1 week) due in part to the time-dependent contraction of the collagen gel-based matrix and the limited cell types and skin tissue components utilized. In contrast, here we describe a new matrix consisting of a silk-collagen composite system that provides long term, stable cultivation with reduced contraction and degradation over time. This matrix supports full thickness skin equivalents which include nerves. The unique silk-collagen composite system preserves cell-binding domains of collagen while maintaining the stability and mechanics of the skin system for long-term culture with silk. The utility of this new composite protein-based biomaterial was demonstrated by bioengineering full thickness human skin systems using primary cells, including nerves and immune cells to establish an HSE with a neuro-immuno-cutaneous system. The HSEs with neurons and hypodermis, compared to in vitro skin-only HSEs controls, demonstrated higher secretion of pro-inflammatory cytokines. Proteomics analysis confirmed the presence of several proteins associated with inflammation across all sample groups, but HSEs with neurons had the highest amount of detected protein due to the complexity of the model. This improved, in vitro full thickness HSE model system utilizes cross-linked silk-collagen as the biomaterial and allows reduced reliance on animal models and provides a new in vitro tissue system for the assessment of chronic responses related to skin diseases and drug discovery., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Stroke, traumatic brain injuries, and other similar conditions often lead to significant loss of functional brain tissue and associated disruption of neuronal signaling. A common strategy for replacing lost neurons is the injection of dissociated neural stem cells or differentiated neurons. However, this method is unlikely to be suitable for replacing large brain cavities, and the resulting distribution of neurons may lack the necessary architecture to support appropriate brain function. Engineered neural tissues may be a viable alternative. Cell death is a prominent concern in neuronal grafting studies, a problem that could be magnified with the transplantation of engineered neural tissues. Here, we examined the effect of one contributor to cell death, acute cerebral inflammation, on neuronal survival after the transplantation of bioengineered constructs based on silk scaffolds. We found evidence of a high degree of inflammation and poor neuronal survival after introducing engineered constructs into the motor cortex of rats. Integrating a corticosteroid (methylprednisolone) into the constructs resulted in significantly improved neuron survival during the acute phase of inflammation. The improved construct survival was associated with decreased markers of inflammation and an anti-inflammatory state of the immune system due to the steroid treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the bone marrow, the interaction of progenitor cells with the vasculature is fundamental for the release of blood cells into circulation. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising protein biomaterial for bone marrow tissue engineering, because of its tunable architecture and mechanical properties, the capacity to incorporate labile compounds without loss of bioactivity and the demonstrated ability to support blood cell formation without premature activation. In this study, we fabricated a custom perfusion chamber to contain a multi-channel lyophilized silk sponge mimicking the vascular network in the bone marrow niche. The perfusion system consisted in an inlet and an outlet and 2 splitters that allowed funneling flow in each single channel of the silk sponge. Computational Fluid Dynamic analysis demonstrated that this design permitted confined flow inside the vascular channels. The silk channeled sponge supported efficient platelet release from megakaryocytes (Mks). After seeding, the Mks localized along SDF-1α functionalized vascular channels in the sponge. Perfusion of the channels allowed the recovery of functional platelets as demonstrated by increased PAC-1 binding upon thrombin stimulation. Further, increasing the number of channels in the silk sponge resulted in a proportional increase in the numbers of platelets recovered, suggesting applicability to scale-up for platelet production. In conclusion, we have developed a scalable system consisting of a multi-channeled silk sponge incorporated in a perfusion chamber that can provide useful technology for functional platelet production ex vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Silk fibroin (SF) is a natural fibrous polymer with strong potential for many biomedical applications. SF has attracted interest in the field of bone tissue engineering due to its extraordinary characteristics in terms of elasticity, flexibility, biocompatibility and biodegradability. However, low osteogenic capacity has limited applications for SF in the orthopedic arena unless suitably functionalized. Hydroxyapatite (HAp) is a well-established bioceramic with biocompatibility and appropriate for constructing orthopedic and dental substitutes. However, HAp ceramics tend to be brittle which can restrict applications in the repair of load-bearing tissues such as bones. Therefore, blending SF and HAp combines the useful properties of both materials as bone constructs for tissue engineering, the subject of this review., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Continuous gradients present at tissue interfaces such as osteochondral systems, reflect complex tissue functions and involve changes in extracellular matrix compositions, cell types and mechanical properties. New and versatile biomaterial strategies are needed to create suitable biomimetic engineered grafts for interfacial tissue engineering. Silk protein-based composites, coupled with selective peptides with mineralization domains, were utilized to mimic the soft-to-hard transition in osteochondral interfaces. The gradient composites supported tunable mineralization and mechanical properties corresponding to the spatial concentration gradient of the mineralization domains (R5 peptide). The composite system exhibited continuous transitions in terms of composition, structure and mechanical properties, as well as cytocompatibility and biodegradability. The gradient silicified silk/R5 composites promoted and regulated osteogenic differentiation of human mesenchymal stem cells in an osteoinductive environment in vitro. The cells differentiated along the composites in a manner consistent with the R5-gradient profile. This novel biomimetic gradient biomaterial design offers a useful approach to meet a broad range of needs in regenerative medicine., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The bone marrow is a soft, spongy, gelatinous tissue found in the hollow cavities of flat and long bones that support hematopoiesis in order to maintain the physiologic turnover of all blood cells. Silk fibroin, derived from Bombyx mori silkworm cocoons, is a promising biomaterial for bone marrow engineering, because of its tunable architecture and mechanical properties, the capacity of incorporating labile compounds without loss of bioactivity and demonstrated ability to support blood cell formation. In this study, we developed a bone marrow scaffold consisting of a modular flow chamber made of polydimethylsiloxane, holding a silk sponge, prepared with salt leaching methods and functionalized with extracellular matrix components. The silk sponge was able to support efficient platelet formation when megakaryocytes were seeded in the system. Perfusion of the chamber allowed the recovery of functional platelets based on multiple activation tests. Further, inhibition of AKT signaling molecule, which has been shown to be crucial in regulating physiologic platelet formation, significantly reduced the number of collected platelets, suggesting the applicability of this tissue model for evaluation of the effects of bone marrow exposure to compounds that may affect platelet formation. In conclusion, we have bioengineered a novel modular system that, along with multi-porous silk sponges, can provide a useful technology for reproducing a simplified bone marrow scaffold for blood cell production ex vivo., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this study, silk fibroin and hyaluronic acid (HA) were enzymatically crosslinked to form biocompatible composite hydrogels with tunable mechanical properties similar to that of native tissues. The formation of di-tyrosine crosslinks between silk fibroin proteins via horseradish peroxidase has resulted in a highly elastic hydrogel but exhibits time-dependent stiffening related to silk self-assembly and crystallization. Utilizing the same method of crosslinking, tyramine-substituted HA forms hydrophilic and bioactive hydrogels that tend to have limited mechanics and degrade rapidly. To address the limitations of these singular component scaffolds, HA was covalently crosslinked with silk, forming a composite hydrogel that exhibited both mechanical integrity and hydrophilicity. The composite hydrogels were assessed using unconfined compression and infrared spectroscopy to reveal of the physical properties over time in relation to polymer concentration. In addition, the hydrogels were characterized by enzymatic degradation and for cytotoxicity. Results showed that increasing HA concentration, decreased gelation time, increased degradation rate, and reduced changes that were observed over time in mechanics, water retention, and crystallization. These hydrogel composites provide a biologically relevant system with controllable temporal stiffening and elasticity, thus offering enhanced tunable scaffolds for short or long term applications in tissue engineering., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Soluble elastin-like peptides (ELPs) can be engineered into a range of physical forms, from hydrogels and scaffolds to fibers and artificial tissues, finding numerous applications in medicine and engineering as "smart polymers". Elastin-like peptides are attractive candidates as a platform for novel biomaterial design because they exhibit a highly tunable response spectrum, with reversible phase transition capabilities. Here, we report the design of the first virtual library of elastin-like protein models using methods for enhanced sampling to study the effect of peptide chemistry, chain length, and salt concentration on the structural transitions of ELPs, exposing associated molecular mechanisms. We describe the behavior of the local molecular structure under increasing temperatures and the effect of peptide interactions with nearest hydration shell water molecules on peptide mobility and propensity to exhibit structural transitions. Shifts in the magnitude of structural transitions at the single-molecule scale are explained from the perspective of peptide-ion-water interactions in a library of four unique elastin-like peptide systems. Predictions of structural transitions are subsequently validated in experiment. This library is a valuable resource for recombinant protein design and synthesis as it elucidates mechanisms at the single-molecule level, paving a feedback path between simulation and experiment for smart material designs, with applications in biomedicine and diagnostic devices., (Copyright © 2017. Published by Elsevier Ltd.)

Fiber-like structures are prevalent in biological tissues, yet quantitative approaches to assess their three-dimensional (3D) organization are lacking. We develop 3D directional variance, as a quantitative biomarker of truly 3D fibrillar organization by extending the directional statistics formalism developed for describing circular data distributions (i.e. when 0° and 360° are equivalent) to axial ones (i.e. when 0° and 180° are equivalent). Significant advantages of this analysis include its time efficiency, sensitivity and ability to provide quantitative readouts of organization over different size scales of a given data set. We establish a broad range of applications for this method by characterizing collagen fibers, neuronal axons and fibroblasts in the context of cancer diagnostics, traumatic brain injury and cell-matrix interactions in developing engineered tissues. This method opens possibilities for unraveling in a sensitive, and quantitative manner the organization of essential fiber-like structures in tissues and ultimately its impact on tissue function., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the field of soft tissue reconstruction, custom implants could address the need for materials that can fill complex geometries. Our aim was to develop a material system with optimal rheology for material extrusion, that can be processed in physiological and non-toxic conditions and provide structural support for soft tissue reconstruction. To meet this need we developed silk based bioinks using gelatin as a bulking agent and glycerol as a non-toxic additive to induce physical crosslinking. We developed these inks optimizing printing efficacy and resolution for patient-specific geometries that can be used for soft tissue reconstruction. We demonstrated in vitro that the material was stable under physiological conditions and could be tuned to match soft tissue mechanical properties. We demonstrated in vivo that the material was biocompatible and could be tuned to maintain shape and volume up to three months while promoting cellular infiltration and tissue integration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The interactions between corneal nerve, epithelium, and stroma are essential for maintaining a healthy cornea. Thus, corneal tissue models that more fully mimic the anatomy, mechanical properties and cellular components of corneal tissue would provide useful systems to study cellular interactions, corneal diseases and provide options for improved drug screening. Here a corneal tissue model was constructed to include the stroma, epithelium, and innervation. Thin silk protein film stacks served as the scaffolding to support the corneal epithelial and stromal layers, while a surrounding silk porous sponge supported neuronal growth. The neurons innervated the stromal and epithelial layers and improved function and viability of the tissues. An air-liquid interface environment of the corneal tissue was also mimicked in vitro, resulting in a positive impact on epithelial maturity. The inclusion of three cell types in co-culture at an air-liquid interface provides an important advance for the field of in vitro corneal tissue engineering, to permit improvements in the study of innervation and corneal tissue development, corneal disease, and tissue responses to environmental factors., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bio-functionalized microfluidic systems were developed based on a silk protein hydrogel elastomeric materials. A facile multilayer fabrication method using gelatin sacrificial molding and layer-by-layer assembly was implemented to construct interconnected, three dimensional (3D) microchannel networks in silk hydrogels at 100 μm minimum feature resolution. Mechanically activated valves were implemented to demonstrate pneumatic control of microflow. The silk hydrogel microfluidics exhibit controllable mechanical properties, long-term stability in various environmental conditions, tunable in vitro and in vivo degradability in addition to optical transparency, providing unique features for cell/tissue-related applications than conventional polydimethylsiloxane (PDMS) and existing hydrogel-based microfluidic options. As demonstrated in the work here, the all aqueous-based fabrication process at ambient conditions enabled the incorporation of active biological substances in the bulk phase of these new silk microfluidic systems during device fabrication, including enzymes and living cells, which are able to interact with the fluid flow in the microchannels. These silk hydrogel-based microfluidic systems offer new opportunities in engineering active diagnostic devices, tissues and organs that could be integrated in vivo, and for on-chip cell sensing systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Silks are appealing materials for numerous biomedical applications involving drug delivery, tissue engineering, or implantable devices, because of their tunable mechanical properties and wide range of physical structures. In addition to the functionalities needed for specific clinical applications, a key factor necessary for clinical success for any implanted material is appropriate interactions with the body in vivo. This review summarizes our current understanding of the in vivo biological responses to silks, including degradation, the immune and inflammatory response, and tissue remodeling with particular attention to vascularization. While we focus in this review on silkworm silk fibroin protein due to the large quantity of in vivo data thanks to its widespread use in medical materials and consumer products, spider silk information is also included if available. Silk proteins are degraded in the body on a time course that is dependent on the method of silk fabrication and can range from hours to years. Silk protein typically induces a mild inflammatory response that decreases within a few weeks of implantation. The response involves recruitment and activation of macrophages and may include activation of a mild foreign body response with the formation of multinuclear giant cells, depending on the material format and location of implantation. The number of immune cells present decreases with time and granulation tissue, if formed, is replaced by endogenous, not fibrous, tissue. Importantly, silk materials have not been demonstrated to induce mineralization, except when used in calcified tissues. Due to its ability to be degraded, silk can be remodeled in the body allowing for vascularization and tissue ingrowth with eventual complete replacement by native tissue. The degree of remodeling, tissue ingrowth, or other specific cell behaviors can be modulated with addition of growth or other signaling factors. Silk can also be combined with numerous other materials including proteins, synthetic polymers, and ceramics to enhance its characteristics for a particular function. Overall, the diverse array of silk materials shows excellent bioresponses in vivo with low immunogenicity and the ability to be remodeled and replaced by native tissue making it suitable for numerous clinical applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophage has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of β-sheet (crystalline) content enabled a short-term release (4-8 h) of either cytokine, with smaller amounts released out to 24 h. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High β-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Silk hydrogels were formulated with anti-vascular endothelial growth factor (anti-VEGF) therapeutics for sustained ocular drug delivery. Using silk fibroin as a vehicle for delivery, bevacizumab-loaded hydrogel formulations demonstrated sustained release of 3 months or greater in experiments in vitro as well as in vivo using an intravitreal injection model in Dutch-belted rabbits. Using both standard dose (1.25mg bevacizumab/50 μL injection) and high dose (5.0mg bevacizumab/50 μL injection) hydrogel formulations, release concentrations were achieved at day 90 that were equivalent or greater than those achieved at day 30 with the positive standard dose control (single injection (50 μL) of 1.25mg bevacizumab solution), which is estimated to be the therapeutic threshold based on the current dosage administration schedule of 1 injection/month. These gels also demonstrated signs of biodegradation after 3 months, suggesting that repeated injections may be possible (e.g., one injection every 3-6 months or longer). Due to its pharmacokinetic and biodegradation profiles, this delivery system may be used to reduce the frequency of dosing for patients currently enduring treatment using bevacizumab or other anti-VEGF therapeutics., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Despite the promise for stem cell-based tissue engineering for regenerative therapy, slow and insufficient vascularization of large tissue constructs negatively impacts the survival and function of these transplanted cells. A combination of channeled porous silk scaffolds and prevascularization with endothelial cells was investigated to test the ability of this tissue engineering strategy to support rapid and extensive vascularization process. We report that hollow channels promote in vitro prevascularization by facilitating endothelial cell growth, VEGF secretion, and capillary-like tube formation. When implanted in vivo, the pre-established vascular networks in the hollow channel scaffolds anastomose with host vessels and exhibit accelerated vascular infiltration throughout the whole tissue construct, which provides timely and sufficient nutrients to ensure the survival of the transplanted stem cells. This tissue engineering strategy can promote the effective application of stem cell-based regeneration to improve future clinical applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In breast and prostate cancer patients, the bone marrow is a preferred site of metastasis. We hypothesized that we could use tissue-engineering strategies to lure metastasizing cancer cells to tissue-engineered bone marrow. First, we generated highly porous 3D silk scaffolds that were biocompatible and amenable to bone morphogenetic protein 2 functionalization. Control and functionalized silk scaffolds were subcutaneously implanted in mice and bone marrow development was followed. Only functionalized scaffolds developed cancellous bone and red bone marrow, which appeared as early as two weeks post-implantation and further developed over the 16-week study period. This tissue-engineered bone marrow microenvironment could be readily manipulated in situ to understand the biology of bone metastasis. To test the ability of functionalized scaffolds to serve as a surrogate niche for metastasis, human breast cancer cells were injected into the mammary fat pads of mice. The treatment of animals with scaffolds had no significant effect on primary tumor growth. However, extensive metastasis was observed in functionalized scaffolds, and the highest levels for scaffolds that were in situ manipulated with receptor activator of nuclear factor kappa-B ligand (RANKL). We also applied this tissue-engineered bone marrow model in a prostate cancer and experimental metastasis setting. In summary, we were able to use tissue-engineered bone marrow to serve as a target or "trap" for metastasizing cancer cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Recombinant spider silks produced in transgenic goat milk were studied as cell culture matrices for neuronal growth. Major ampullate spidroin 1 (MaSp1) supported neuronal growth, axon extension and network connectivity, with cell morphology comparable to the gold standard poly-lysine. In addition, neurons growing on MaSp1 films had increased neural cell adhesion molecule (NCAM) expression at both mRNA and protein levels. The results indicate that MaSp1 films present useful surface charge and substrate stiffness to support the growth of primary rat cortical neurons. Moreover, a putative neuron-specific surface binding sequence GRGGL within MaSp1 may contribute to the biological regulation of neuron growth. These findings indicate that MaSp1 could regulate neuron growth through its physical and biological features. This dual regulation mode of MaSp1 could provide an alternative strategy for generating functional silk materials for neural tissue engineering., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II, β-sheet content via "film-spinning." Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in vitro and in vivo pharmacokinetic data (in a female Sprague-Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in vitro and in vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In vivo results highlighted a strong in vitro-in vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and β-sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Limitations of current clinical methods for bone repair continue to fuel the demand for a high strength, bioactive bone replacement material. Recent attempts to produce porous scaffolds for bone regeneration have been limited by the intrinsic weakness associated with high porosity materials. In this study, ceramic scaffold fabrication techniques for potential use in load-bearing bone repairs have been developed using naturally derived silk from Bombyx mori. Silk was first employed for ceramic grain consolidation during green body formation, and later as a sacrificial polymer to impart porosity during sintering. These techniques allowed preparation of hydroxyapatite (HA) scaffolds that exhibited a wide range of mechanical and porosity profiles, with some displaying unusually high compressive strength up to 152.4 ± 9.1 MPa. Results showed that the scaffolds exhibited a wide range of compressive strengths and moduli (8.7 ± 2.7 MPa to 152.4 ± 9.1 MPa and 0.3 ± 0.1 GPa to 8.6 ± 0.3 GPa) with total porosities of up to 62.9 ± 2.7% depending on the parameters used for fabrication. Moreover, HA-silk scaffolds could be molded into large, complex shapes, and further machined post-sinter to generate specific three-dimensional geometries. Scaffolds supported bone marrow-derived mesenchymal stem cell attachment and proliferation, with no signs of cytotoxicity. Therefore, silk-fabricated HA scaffolds show promise for load bearing bone repair and regeneration needs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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 © 2014 Elsevier Ltd. All rights reserved.)

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., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

The goal of this study was to explore quantitative assessments of mineralized silk protein biomaterial films by co-cultures of human mesenchymal stem cell-derived osteoblasts and human acute monocytic leukemia cell line-derived osteoclasts during long-term culture (8-32 weeks). The remodeled films were quantitatively assessed using three different techniques during this extended cultivation to provide more comprehensive insight into the impact of co-cultures on surface remodeling. Scanning electron microscopy (SEM) with three dimensional surface reconstructions was used to quantitatively determine various surface morphological features and measures of roughness indicative of remodeling by the cells. Additionally, reconstructed surfaces were converted to depth images for Fourier analysis to quantify the potential fractal organization of biomineralization. The long-term remodeled films were also imaged using confocal reflectance microscopy and micro-computed tomography (micro-CT) to further quantify morphological changes. Films remodeled in co-culture demonstrated increased roughness parameters, fractal organization, and volume compared to films remodeled by osteoblasts alone. The combination of these techniques to quantify remodeling of mineralized protein films shows promise for quantifying processes related to mineralized surfaces., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Peripheral nervous system injuries result in a decreased quality of life, and generally require surgical intervention for repair. Currently, the gold standard of nerve autografting, based on the use of host tissue such as sensory nerves is suboptimal as it results in donor-site loss of function and requires a secondary surgery. Nerve guidance conduits fabricated from natural polymers such as collagen are a common alternative to bridge nerve defects. In the present work, tendon sections derived through a process named bioskiving were studied for their potential for use as a substrate to fabricate nerve guidance conduits. We show that cells such as rat Schwann cells adhere, proliferate, and align along the fibrous tendon substrate which has been shown to result in a more mature phenotype. Additionally we demonstrate that chick dorsal root ganglia explants cultured on the tendon grow to similar lengths compared to dorsal root ganglia cultured on collagen gels, but also grow in a more oriented manner on the tendon sections. These results show that tendon sections produced through bioskiving can support directional nerve growth and may be of use as a substrate for the fabrication of nerve guidance conduits., (Copyright © 2014 Elsevier Ltd. All rights reserved.)