Mori Silkworm Research Articles

Biological Effect Evaluation of Different Sized Titanium Dioxide Nanoparticles Using Bombyx mori (Silkworm) as a Model Animal.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in various disciplines, and it is imperative to evaluate their safety in the environment. In this paper, Bombyx mori (silkworm) was used as a model organism to evaluate the biological effects of different sized TiO2 NPs, taking into consideration their effect on the larval growth, cocoon shell weight, tissues, and silk produced. The effect of the different sized TiO2 NPs on the larval and cocoon shell weight was dose-dependent. The highest accumulation of titanium (Ti) following a modified TiO2 NPs-treated mulberry diet was observed in the midgut. The expression of the light chain fibroin (FIBL) was three times higher in 0.33 g TiO2 NPs-treated silk gland after 96h. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analysis demonstrated that TiO2 NPs-treated silk fiber (TiSF) exhibited a diminutive decrease in silk fiber (SF) crystallization and β-sheet compared to the control SF, respectively. The tensile tests of SF from silkworm fed with 0.03g of 25nm TiO2 NPs were significantly improved when compared to the control SF. Significant changes in the surface morphology and thermal stability of SF were observed. The antimicrobial activity of TiSF was investigated against Pseudomonas aeruginosa and Staphylococcus aureus, with ciprofloxacin-treated SF acting as a control. It was documented that 0.09g of 60nm TiSF was most effective against P. aeruginosa at a zone of inhibition (ZOI) of 21.06mm when compared with the control SF which recorded a ZOI of 17.19mm. This study highlighted a different approach in evaluating the biological effects of TiO2 NPs using the silkworm as a model and assessing their impact on the silk intrinsic property, which will be effective in biotechnology applications.

From Mesoscopic Functionalization of Silk Fibroin to Smart Fiber Devices for Textile Electronics and Photonics.

Bombyx mori silk fibers exhibit significant potential for applications in smart textiles, such as fiber sensors, fiber actuators, optical fibers, and energy harvester. Silk fibroin (SF) from B. mori silkworm fibers can be reconstructed/functionalized at the mesoscopic scale during refolding from the solution state into fibers. This facilitates the mesoscopic functionalization by engaging functional seeds in the refolding of unfolded SF molecules. In particular, SF solutions can be self‐assembled into regenerated fiber devices by artificial spinning technologies, such as wet spinning, dry spinning, microfluidic spinning, electrospinning, and direct writing. Meso‐functionalization manipulates the SF property from the mesoscopic scale, transforming the original silk fibers into smart fiber devices with smart functionalities, such as sensors, actuators, optical fibers, luminous fibers, and energy harvesters. In this review, the progress of mesoscopic structural construction from SF materials to fiber electronics/photonics is comprehensively summarized, along with the spinning technologies and fiber structure characterization methods. The applications, prospects, and challenges of smart silk fibers in textile devices for wearable personalized healthcare, self‐propelled exoskeletons, optical and luminous fibers, and sustainable energy harvesters are also discussed.

Ecopoetic Antinomies: Inscription and Voice in Jen Bervin’s Silk Poems

Text means Tissue; but whereas hitherto we have always taken this tissue as a product, a ready-made veil, behind which lies, more or less hidden, meaning (truth), we are now emphasizing, in the tissue, the generative idea that the text is made, is worked out in a perpetual interweaving; lost in this tissue—this texture—the subject unmakes himself, like a spider dissolving in the constructive secretions of its web. Roland Barthes, The Pleasure of the Text, 64 The multi-formatted poem-object that Jen Bervin calls Silk Poems (2010–20) features an exceptionally charismatic speaker. “ok/embarrassedtosay/inmygreatexcitement/ifellasleep” it confesses upon waking from a mid-poem nap (61). From the urgent materiality of its growing body, the poem’s Bombyx Mori silkworm–speaker addresses the imagined human recipient of a biomedical liquid silk sensor—designed to be implanted inside the human body during illness—made from the silk it uses to weave its cocoon: “ithought/youshould/know/howitis/with/the/creatures/whomadethis,” it avers (1). What follows is a series of narrative scenes, diversions, distractions, and interruptions, as the silkworm weaves together the complex layers of cultural and linguistic history embedded in the materiality of silk from within its own bodily growth as it passes through “instars,” stages of insect development between moltings of skin, from birth to silk production to death. Both expansive in its knowledge of literary and silk history and intimately enmeshed within the demands of its body (“i’msohungryi/shuckmyskin/anddevour/it whole,” it remarks), the silkworm–speaker enacts and transcribes the material history and conditions of its existence (62). In doing so, the silkworm shows how silk’s historical and literary lives are entwined with its material ones. Bervin would seem to propose that the junction between writing and materiality is an embedded property of language: “The page is not quiet,” she writes, “It has been written endlessly” (“Text from Textile” 87).

Structure of the Gamma Ray Irradiation-Curable Liquid Silk 3D Scaffold with Cell-Adhesive Property

We attempted to produce three dimensional (3D) aero-sponges using liquid silk (LS) extracted from the silk glands of fully grown larvae of Bombyx mori silkworms by combining an ice-crystal-sublimation method and a water-annealing treatment. The pore structure of the 3D LS-aero-sponge was constructed by the branched fibrous substrates and the films with small holes. It was assumed that the branched fibrous structure was mainly comprised of fibroin whereas the films were comprised of the fibroin interacting with the sericin fractions. Most of the WAXD peaks from the as-molded 3D LS-aero-sponge could be assigned to the silk1 crystal diffraction peaks of fibroin. The silk1 crystal modification of fibroin formed in the course of the production of the 3D LS-aero-sponge could bring about a good thermal shrinkproof property. Furthermore, a gamma ray irradiation treatment applied prior to the cell proliferation tests of the 3D LS-aero-sponges was found to be effective to form cross-linking in the sericin fractions. As a result, the yield stress in the compression tests of the 3D LS-aero-sponges was increased. The 3D LS-aero-sponge exhibited a good living cell proliferation properly in the culture tests using NH3T3 cells (Swiss albino mice embryonic fibroblasts) although that of the 2D LS-cast films was very poor. We suggest the good cell proliferation property of the 3D LS-aero-sponge was due to the continuous branched fibrous structure comprised of fibroin.

Mesoscale structure development reveals when a silkworm silk is spun

Silk fibre mechanical properties are attributed to the development of a multi-scale hierarchical structure during spinning. By careful ex vivo processing of a B. mori silkworm silk solution we arrest the spinning process, freezing-in mesoscale structures corresponding to three distinctive structure development stages; gelation, fibrilization and the consolidation phase identified in this work, a process highlighted by the emergence and extinction of ‘water pockets’. These transient water pockets are a manifestation of the interplay between protein dehydration, phase separation and nanofibril assembly, with their removal due to nanofibril coalescence during consolidation. We modeled and validated how post-draw improves mechanical properties and refines a silk’s hierarchical structure as a result of consolidation. These insights enable a better understanding of the sequence of events that occur during spinning, ultimately leading us to propose a robust definition of when a silkworm silk is actually ‘spun’.

Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pébrine disease in Bombyx mori silkworm, based on the β-tubulin gene

“Pébrine” is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the β-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.

Synergistic Neuroprotective Effects of Mature Silkworm and Angelica gigas Against Scopolamine-Induced Mild Cognitive Impairment in Mice and H2O2-Induced Cell Death in HT22 Mouse Hippocampal Neuronal Cells.

We previously reported that mature Bombyx mori silkworm (SW) ameliorated scopolamine (Sco)-induced amnesia, and Angelica gigas (AG) prevented cognitive impairment. SW is known for its gastroprotective effects such as improving liver function and alleviating the effects of Parkinson's disease. AG is known for its neuroprotective effects and for lowering the effects of low-density lipoprotein cholesterol. However, the neuroprotective effect of combined SW and AG (SWA-1) treatment and the underlying molecular mechanism by which SWA-1 regulates neurodegenerative diseases remains unclear. We evaluated the neuroprotective effect of SWA-1 against Sco-induced mild cognitive impairment in mice and H2O2-induced cell death in HT22 mouse hippocampal neuronal cells and elucidated the underlying molecular mechanism. Morris water maze and Y-maze tests were performed to examine the learning and memory abilities of mice. The underlying molecular mechanism was investigated by using western blotting. We demonstrated that SWA-1 significantly protects against H2O2-induced cell death in HT22 mouse hippocampal neuronal cells. SWA-1 also significantly reversed Sco-induced spatial learning and memory impairment. Specifically, SWA-1 upregulates the protein levels of phosphorylated extracellular signal-related kinase (Erk1/2) and phosphorylated p38 MAP kinase (p38). SWA-1 remarkably decreased the apoptotic index Bax/Bcl2 expression in the hippocampus of Sco-treated mice. Our results suggest that SWA-1 may be administered as alternative therapy for cognitive impairment and neurodegenerative diseases and should be studied further in human trials.

Chemical, Thermal, Time, and Enzymatic Stability of Silk Materials with Silk I Structure.

The crystalline structure of silk fibroin Silk I is generally considered to be a metastable structure; however, there is no definite conclusion under what circumstances this crystalline structure is stable or the crystal form will change. In this study, silk fibroin solution was prepared from B. Mori silkworm cocoons, and a combined method of freeze-crystallization and freeze-drying at different temperatures was used to obtain stable Silk I crystalline material and uncrystallized silk material, respectively. Different concentrations of methanol and ethanol were used to soak the two materials with different time periods to investigate the effect of immersion treatments on the crystalline structure of silk fibroin materials. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman scattering spectroscopy (Raman), Scanning electron microscope (SEM), and Thermogravimetric analysis (TGA) were used to characterize the structure of silk fibroin before and after the treatments. The results showed that, after immersion treatments, uncrystallized silk fibroin material with random coil structure was transformed into Silk II crystal structure, while the silk material with dominated Silk I crystal structure showed good long-term stability without obvious transition to Silk II crystal structure. α-chymotrypsin biodegradation study showed that the crystalline structure of silk fibroin Silk I materials is enzymatically degradable with a much lower rate compared to uncrystallized silk materials. The crystalline structure of Silk I materials demonstrate a good long-term stability, endurance to alcohol sterilization without structural changes, and can be applied to many emerging fields, such as biomedical materials, sustainable materials, and biosensors.

Natural silk fiber microcarriers for cell culture

The development of effective and versatile microcarriers is a pressing issue in tissue engineering and regenerative medicine. The objective of this wor k is to create biocompatible fiber microparticles from the cocoons of the Bombyx mori silkworm, and to study their structure and biological properties. Materials and methods . In obtaining microparticles, the Bombyx mori cocoons washed from sericin were cryo-milled in liquid nitrogen. The structure of the resulting microparticles was analyzed via scanning electron microscopy. The cytotoxicity of the obtained fibers was assessed using MTT-cell culture assay of 3T3 mouse fibroblasts. Cell adhesion analysis was performed using the Hep-G 2 human hepatocarcinoma cell line. Cell visualization was performed by staining the nuclei with DAPI fluorescent dye. Results . Natural silk microparticles were obtained in the form of cylindrical fibers with 200–400 μm average length and 15 μm diameter. It was shown that the surface of the resulting microparticles has a rough relief; no pores were found. The microparticles are non-toxic for 3T3 mouse fibroblasts, they maintain a high level of adhesion by human hepatocellular carcinoma HepG 2 cells . Conclusion. The method developed by us for fabrication of biocompatible silk fibroin microparticles in the form of fibers without using toxic reagents and significant time costs is promising for cell cultivation and delivery to the damaged area for tissue and organ regeneration.

Natural silk-composite enabled versatile robust triboelectric nanogenerators for smart applications

Strategies to maximize the surface charge density across triboelectric layers while protecting it from humidity are crucial in employing triboelectric nanogenerators (TENGs) for commercial/real-time applications. Herein, for the first time, we propose the utility of crystalline silk microparticles (SMPs) to improve the surface charge density in materials like polyvinyl alcohol to realise its applicability for TENG devices. Moreover, these SMPs are extracted from discarded Bombyx mori silkworm cocoons by facile, inexpensive, and single-step alkaline-hydrolysis treatment. We examine the performance of these composites with counter-materials composed of waste PTFE plastic cups to show reuse in recycled products. The processing cost of TENG developed from recycled materials is not only low but eco-friendly. The TENG performance as a function of the concentration of SMPs is investigated and compared with the composite's work-function and surface-potentials, with the distance-dependent electric field theoretical model employed to optimize the performance. Consequently, the optimized TENG exhibits maximum output voltage, current, charge, and power density of ∼280 V, 17.3 μA, 32.5 nC, and 14.4 W·m−2, respectively, creating a highly competitive energy harvester that can conform to the rigorous needs of wearables and mobile applications. Furthermore, the fully packaged silicone rubber device protects it from humidity and enables the device utility for practical applications with a soft, comfortable, and skin-friendly interface.

Assessment of diseases in bombyx mori silkworm – A survey

India, the second highest producer of silk in the world as well as the largest consumer. The demand for silk is rising day by day, on the other hand, there is also rise in production in parallel. According to the annual report 2020 of Ministry of Textiles, Government of India, there is a huge rise in the production of silk and it is rising year by year. During 2018-20, total production of silk in India was about 35468 metric tons. Although there is so much demand as well as supply, the farmers are facing lot of problems because of silkworm being borne to various kinds of diseases. Although government is organizing various programs to provide knowledge to the farmers, yet the loss of crop is not controlled so far as expected. This paper reflects the survey on various types of silkworm used to produce silk, the threat of diseases, factors that influence the diseases, control of diseases, treatments and some technical over-view on utilization of Image processing techniques to overcome these problems.

Oral Vaccination of Grass Carp (Ctenopharyngodon idella) with Baculovirus-Expressed Grass Carp Reovirus (GCRV) Proteins Induces Protective Immunity against GCRV Infection.

The grass carp reovirus (GCRV) causes severe hemorrhagic disease with high mortality and leads to serious economic losses in the grass carp (Ctenopharyngodon idella) industry in China. Oral vaccine has been proven to be an effective method to provide protection against fish viruses. In this study, a recombinant baculovirus BmNPV-VP35-VP4 was generated to express VP35 and VP4 proteins from GCRV type Ⅱ via Bac-to-Bac baculovirus expression system. The expression of recombinant VP35-VP4 protein (rVP35-VP4) in Bombyx mori embryo cells (BmE) and silkworm pupae was confirmed by Western blotting and immunofluorescence assay (IFA) after infection with BmNPV-VP35-VP4. To vaccinate the grass carp by oral route, the silkworm pupae expressing the rVP35-VP4 proteins were converted into a powder after freeze-drying, added to artificial feed at 5% and fed to grass carp (18 ± 1.5 g) for six weeks, and the immune response and protective efficacy in grass carp after oral vaccination trial was thoroughly investigated. This included blood cell counting and classification, serum antibody titer detection, immune-related gene expression and the relative percent survival rate in immunized grass carp. The results of blood cell counts show that the number of white blood cells in the peripheral blood of immunized grass carp increased significantly from 14 to 28 days post-immunization (dpi). The differential leukocyte count of neutrophils and monocytes were significantly higher than those in the control group at 14 dpi. Additionally, the number of lymphocytes increased significantly and reached a peak at 28 dpi. The serum antibody levels were significantly increased at Day 14 and continued until 42 days post-vaccination. The mRNA expression levels of immune-related genes (IFN-1, TLR22, IL-1β, MHC I, Mx and IgM) were significantly upregulated in liver, spleen, kidney and hindgut after immunization. Four weeks post-immunization, fish were challenged with virulent GCRV by intraperitoneal injection. The results of this challenge study show that orally immunized group exhibited a survival rate of 60% and relative percent survival (RPS) of 56%, whereas the control group had a survival rate of 13% and RPS of 4%. Taken together, our results demonstrate that the silkworm pupae powder containing baculovirus-expressed VP35-VP4 proteins could induce both non-specific and specific immune responses and protect grass carp against GCRV infection, suggesting it could be used as an oral vaccine.

Self-Assembly of Bombyx mori Silk Fibroin.

Silk fibroin from Bombyx mori (silkworm) distinguishes for its unique mechanical performance, controllable degradation rates, and easily large-scale production, making it attractive models for a variety of biomaterial design. These outstanding properties of silk fibroin originate from its unique modular composition of silk proteins. To exploit the structure-function relationship and fabricate silk fibroin-based biomaterials, comprehensive strategies to uncover assembly behaviors of fibrous proteins are necessary. This chapter describes methods to produce regenerated silk fibroin protein from Bombyx mori silk and their self-assembly strategies. This could provide insight into the fabrication of various silk fibroin-based biomaterials, such as hydrogels, tubes, sponges, fibers, microspheres, and diverse thin film patterns, which can be used for textiles, electronics and optics, environmental engineering, and biomedical applications.

Effects of pyriproxyfen exposure on damage to midgut and related gene expressions in the Bombyx mori silkworm

Effects of pyriproxyfen exposure on damage to midgut and related gene expressions in the Bombyx mori silkworm

Silk Protein Paper with In Situ Synthesized Silver Nanoparticles.

Silver nanoparticles (AgNPs) are in situ synthesized for the first time on microfibrillated silk (MFS) exfoliated from domesticated Philosamia cynthia ricini (eri) and Bombyx mori (mulberry) silkworm silk fibers. The process is rapid (hours time), does not rely on harmful chemicals, and produces robust and flexible AgNPs coated MFS (MFS-AgNPs) protein papers with excellent handling properties. None of these can be achieved by approaches used in the past to fabricate AgNPs silk systems. MFS bonds the AgNPs strongly, providing good support and stabilization for the NPs, leading to strong wash fastness. The mechanical properties of the MFS-AgNPs papers largely do not change compared to the MFS papers without nanoparticles, except for some higher concentration of AgNPs in the case of mulberry silk. The improved tensile properties of eri silk papers with or without AgNPs compared to mulberry silk papers can be attributed to the higher degree of fibrillation achieved in eri silk and its inherent higher ductility. MFS-AgNPs from eri silk also exhibit strong antibacterial activity. This study provides the basis for the development of smart protein papers based on silk fiber and functional nanomaterials.

Crystallization Behavior of Bombyx mori Silkworm Fibroin in Liquid Silk in Static and Flow States: Influence of the Interactions of the Sericin Fractions with the Fibroin

The self-assembling behavior of Bombyx mori silkworm fibroin was investigated focusing on the interaction of the two types of silkworm sericin fractions (P, M) with the fibroin in the static and flow states. The dissociation of the sericin M fraction from the fibroin allowed the fibroin to form the silk2 crystal guided by the sericin P fraction still interacting with the fibroin. Water annealing in room temperature water, or the enzymatic hydrolysis caused by the contamination by fibroinase, weakened the interaction between the fibroin and the sericin M fraction. Thus, the interaction of the sericin P fraction with the fibroin was considered to be more stable than that of the sericin M fraction. As a result all of the entities that assembled in the flow state gave almost similar WAXD intensity curves although the coagulation conditions were changed at every trial. The morphology of the entities obtained in the flow state became film-like when a two-dimensional interface was generated between the fibroin and the free sericin molecules. On the other hand, it became fibrous when the fibroin interacted with a new protein fraction (a minor protein fraction that can cause the solution of fibroin into water) instead of the sericin fractions.

Crosslinked alginate/sericin particles for bioadsorption of ytterbium: Equilibrium, thermodynamic and regeneration studies

Sericin is a soluble globular protein, present in Bombyx mori silkworm cocoons. Sericin's properties can be improved to expand its application by producing blends with other substances, such as alginate polysaccharide and crosslinking agent poly(vinyl alcohol). This study evaluates the use of alginate and sericin particles chemically crosslinked with poly(vinyl alcohol) (SAPVA) for batch bioadsorption of rare-earth element ytterbium from aqueous medium. The equilibrium study showed that the maximum bioadsorption capacity for ytterbium was 0.642 mmol/g at 55 °C. Equilibrium data fit both Langmuir and Dubinin-Radushkevich models. The estimation of thermodynamic parameters showed that there was an increase in the entropy change, and that the bioadsorption process is endothermic and spontaneous. Characterization analyzes revealed that SAPVA particles, even after ytterbium bioadsorption, showed spherical shape, homogeneous composition, amorphous structure, low surface area, macropores, and low porosity. After the first regeneration cycle, the amount of captured ytterbium ions showed a slight increase (about 0.01 mmol/g) and calcium ions were completely released by SAPVA particles. Bioadsorbent particles separated selectively ytterbium from synthetic effluent containing different toxic metal ions. These results show that the SAPVA particles can be used as an effective bioabsorbent to remove and recover ytterbium from wastewater.

Characterization of Electrospun Silk Fibroin Scaffolds for Bone Tissue Engineering: A Review

Silk Fibroin (SF) is a natural polymer obtained from the Bombyx mori silkworm. It has been used in bone tissue engineering thanks to its favorable biocompatibility, adhesion, low biodegradability, and tensile strength properties. Electrospinning is a technique to develop nanofibers. It uses high voltages to convert polymer solutions into porous nanostructured scaffolds with a good ratio between superficial area and volume. In this paper, we examine the effect of the electrospinning parameters on fiber morphology once the spun fibers have been treated. In addition, we present different physicochemical characterizations of electrospun SF scaffolds such as their morphology (via Scanning Electron Microscopic—SEM—), crystalline structure (via Fourier Transform Infrared—FTIR—spectroscopy and X-Ray Diffraction—XRD—), thermal characteristics (via Differential Scanning Calorimetry—DSC—and Thermogravimetric Analysis—TGA—), and mechanical properties (tensile strength). Finally, we discuss the potential applications and impacts of electrospun SF in bone tissue engineering and future research trends.

Using Eukaryotic Expression Systems to Generate Human α1,3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells.

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.

Silk fibroin reactive inks for 3D printing crypt-like structures

A reactive silk fibroin ink formulation designed for extrusion three-dimensional (3D) printing of protein-based hydrogels at room temperature is reported. This work is motivated by the need to produce protein hydrogels that can be printed into complex shapes with long-term stability using extrusion 3D printing at ambient temperature without the need for the addition of nanocomposites, synthetic polymers, or sacrifical templates. Silk fibroin from the Bombyx mori silkworm was purified and synthesized into reactive inks by enzyme-catalyzed dityrosine bond formation. Rheological and printing studies showed that tailoring the peroxide concentration in the reactive ink enables the silk to be extruded as a filament and printed into hydrogel constructs, supporting successive printed layers without flow of the construct or loss of desired geometry. To enable success of longer-term in vitro studies, 3D printed silk hydrogels were found to display excellent shape retention over time, as evidenced by no change in construct dimensions or topography when maintained for nine weeks in culture medium. Caco-2 (an intestinal epithelial cell line) attachment, proliferation, and tight junction formation on the printed constructs was not found to be affected by the geometry of the constructs tested. Intestinal myofibroblasts encapsulated within reactive silk inks were found to survive shearing during printing and proliferate within the hydrogel constructs. The work here thus provides a suitable route for extrusion 3D printing of protein hydrogel constructs that maintain their shape during printing and culture, and is expected to enable longer-term cellular studies of hydrogel constructs that require complex geometries and/or varying spatial distributions of cells on demand via digital printing.