Silk Fibroin Hydrogels Research Articles

Biomimetic silk fibroin and xanthan gum blended hydrogels for connective tissue regeneration

Combination of naturally occurring materials instead of chemically synthesized products has always been an attractive proposition in the field of tissue engineering. In this study, silk fibroin (SF) and xanthan gum(XG) were physically crosslinked to form biocompatible hydrogels. SF/XG hydrogels were prepared using ultrasonication, which induces β-sheets from random coils in SF solution and allows entrapment of heated XG chains homogeneously in the SF network. It is a novel way of blending SF and XG polymers which avoids the usage of chemical crosslinkers. SF/XG blended solutions were used at different ratios for the hydrogel formation. Scanning electron microscopy (SEM) and micro-computed tomography (MCT) were used for morphological analysis of the interconnected network and porosity of the scaffolds, respectively. Rheological studies were performed to understand the changes in mechanical properties due to the incorporation of XG into SF hydrogels. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of SF and XG moieties in the blend scaffolds. Additionally, thermal Analysis (TGA & DSC) established the homogenous mixture and presence of XG in the SF network without any phase separation. Furthermore, the MTT assay demonstrates the cytocompatibility of scaffolds using L929 fibroblast cells. Thus, fabricated SF/XG scaffolds could mimic natural cartilage ECM by exhibiting enhanced water swelling capacity and suitable porosity along with its cytocompatible studies, indicating its potential application in soft tissue engineering.

First steps for the development of silk fibroin-based 3D biohybrid retina for age-related macular degeneration (AMD)

Age-related macular degeneration is an incurable chronic neurodegenerative disease, causing progressive loss of the central vision and even blindness. Up-to-date therapeutic approaches can only slow down he progression of the disease. Objective. Feasibility study for a multilayered, silkfibroin-based, 3D biohybrid retina. Approach. Fabrication of silk fibroin-based biofilms; culture of different types of cells: retinal pigment epithelium, retinal neurons, Müller and mesenchymal stem cells ; creation of a layered structure glued with silk fibroin hydrogel. Main results. In vitro evidence for the feasibility of layered 3D biohybrid retinas; primary culture neurons grow and develop neurites on silk fibroin biofilms, either alone or in presence of other cells cultivated on the same biomaterial; cell organization and cellular phenotypes are maintained in vitrofor the seven days of the experiment. Significance. 3D biohybrid retina can be built using silksilkworm fibroinfilms and hydrogels to be used in cell replacement therapy for AMD and similarretinal neurodegenerative diseases.

High-Strength and High-Toughness Silk Fibroin Hydrogels: A Strategy Using Dynamic Host-Guest Interactions.

Natural polymer-based hydrogels attract great attention because of their inherent biocompatibility and controllable biodegradability. However, the broad applications of these hydrogels require a combination of high mechanical strength, high toughness, fatigue resistance, as well as self-healing. The integration of this combination into one natural polymer-based hydrogel remains challenging. Here, a molecular design strategy was proposed to fabricate mechanically robust silk fibroin-based hydrogels using host-guest interactions. Silk fibroin molecules was chemically modified with cholesterol (Chol, guest) or β-cyclodextrin (β-CD, host), and host-guest interaction between Chol and β-CD moieties drove the supramolecular assemblies of hydrogels. The dissociation/reassociation behavior of host-guest complexation, serving as sacrificial bonds, endowed hydrogels with effective energy dissipation and rapid self-healing ability. The prepared silk fibroin-based hydrogels exhibited high mechanical strength, high toughness, and remarkable fatigue resistance, superior to conventional silk fibroin hydrogels. Moreover, due to reversible host-guest interactions, hydrogels achieved facile functional recovery after damage without any external stimuli. This design strategy provides an avenue to develop natural polymer-based materials with robust mechanical properties, thus broadening current hydrogel applications.

Programing Performance of Silk Fibroin Superstrong Scaffolds by Mesoscopic Regulation among Hierarchical Structures.

To design higher-strength natural scaffold materials, wool keratin (WK) rich in α-helix structures is used as a well-defined foreign substrate, which induces the formation of β-crystallites in silk fibroin (SF). Consequently, the macroscopic properties of silk materials (such as the rheological properties of SF hydrogels and the mechanical properties of stents) can be manipulated by governing the change in the hierarchical mesoscopic structure of silk materials. In this work, by monitoring the structure and morphology in the SF gel process, the mechanism of the effect of keratin on SF network formation was speculated, which was further used to design ultra-high-strength protein scaffolds. It has been confirmed that WK accelerates the gelation of SF by reducing the multistep nucleation barrier and increasing the primary nucleation sites, and then establishing a high-density SF domain network. The modulus of the protein composite scaffold prepared by this facile strategy can reach 11.55 MPa, and the MC-3T3 cells can grow well on the scaffold surface. The results suggest that freeze-dried biocompatible SF-based scaffolds are potential candidates for bone tissue engineering.

Dual-functional liposomes for curcumin delivery and accelerating silk fibroin hydrogel formation.

The administration of a drug-loaded implantable hydrogel at the tumor site after surgical resection is a viable approach to prevent the local recurrence or metastasis. Dimyristoyl glycerophosphorylglycerol (DMPG)-based liposomes were developed for inducing the rapid gelation of silk fibroin (SF) and delivering an anticancer drug, curcumin. Curcumin was loaded in the liposomes and the stability of curcumin was enhanced. The gelation time of liposome-induced SF hydrogels ranged from 3min to more than 6h. The biological activity of liposome-SF hydrogels was evaluated in vitro using L929 fibroblasts and MDA-MB-231 breast cancer cells. The release of curcumin can inhibit the growth of cancer cells. Both cells cultured on the surface of the hydrogels loaded with curcumin displayed low cell survival due to the combination of low cell attachment and cytotoxicity of curcumin. Liposome-SF hydrogels show potential as a sealant administered at the tumor site to eliminate residual cancer cells after tumor removal.

4D-bioprinted silk hydrogels for tissue engineering

Recently, four-dimensional (4D) printing is emerging as the next-generation biofabrication technology. However, current 4D bioprinting lacks biocompatibility or multi-component printability. In addition, suitable implantable targets capable of applying 4D bioprinted products have not yet been established, except theoretical and in vitro study. Herein, we describe a cell-friendly and biocompatible 4D bioprinting system including more than two cell types based on digital light processing (DLP) and photocurable silk fibroin (Sil-MA) hydrogel. The shape changes of 3D printed bilayered Sil-MA hydrogels were controlled by modulating their interior or exterior properties in physiological conditions. We used finite element analysis (FEA) simulations to explore the possible changes in the complex structure. Finally, we made trachea mimetic tissue with two cell types using this 4D bioprinting system and implanted it into a damaged trachea of rabbit for 8 weeks. The implants were integrated with the host trachea naturally, and both epithelium and cartilage were formed at the predicted sites. These findings demonstrate that 4D bioprinting system could make tissue mimetic scaffold biologically and suggest the potential value of the 4D bioprinting system for tissue engineering and the clinical application.

Stretchable silk fibroin hydrogels

Hydrogels derived from silk fibroin (SF) are attractive soft materials in biomedical applications such as drug delivery and tissue engineering. However, SF hydrogels reported so far are generally brittle in tension limiting their load-bearing applications. We present here a novel strategy for preparing stretchable SF hydrogels by incorporating flexible polymer chains into the brittle SF network, which strengthen the interconnections between SF globules. We included N, N-dimethylacrylamide (DMAA) monomer and ammonium persulfate initiator into an aqueous SF solution containing a diepoxide cross-linker to in situ generate flexible poly (N,N-dimethylacrylamide) (PDMAA) chains. Moreover, instead of SF, methacrylated SF was used for the gel preparation to create an interconnected SF/PDMAA network. The free-radical polymerization of DMAA leads to the formation of PDMAA chains interconnecting globular SF molecules via their pendant vinyl groups. Incorporation of 2 w/v% DMAA into the SF network turns the brittle hydrogel into a stretchable one sustaining up to 370% elongation ratio. The mechanical properties of SF hydrogels could be adjusted by the amount of PDMAA incorporated into the SF network. The stretchable and tough SF hydrogels thus developed are suitable as a scaffold in tissue engineering and offer an advantage as a biomaterial over other SF-based biomaterials.

Injectable ferrimagnetic silk fibroin hydrogel for magnetic hyperthermia ablation of deep tumor

Due to the well-recognized biocompatibility, silk fibroin hydrogels have been developed for biomedical applications including bone regeneration, drug delivery and cancer therapy. For the treatment of cancer, silk-based photothermal agents exhibit the high photothermal conversion efficiency, but the limited light penetration depth of photothermal therapy restricts the treatment of some tumors in deep positions, such as liver tumor and glioma. To provide an alternative strategy, here we developed an injectable magnetic hydrogel based on silk fibroin and iron oxide nanocubes (IONCs). The as-prepared ferrimagnetic silk fibroin hydrogel could be easily injected through a syringe into tumor, especially rabbit hepatocellular carcinoma in deeper positions using ultrasound-guided interventional treatment. Compared with photothermal agents, the embedded IONCs endowed the ferrimagnetic silk fibroin hydrogel with remote hyperthermia performance under an alternating magnetic field, resulting in the effective magnetic hyperthermia of deep tumors including subcutaneously implanted tumor model in Balb/c mouse after the coverage of a fresh pork tissue and orthotopic transplantation liver tumor in rabbit. Furthermore, due to the confinement of IONCs in silk fibroin hydrogel, the undesired thermal damage toward normal tissue could be avoided compared with directly administrating monodispersed magnetic nanoparticles.

Sustained Release of TPCA-1 from Silk Fibroin Hydrogels Preserves Keratocyte Phenotype and Promotes Corneal Regeneration by Inhibiting Interleukin-1β Signaling.

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies that exists. Many studies focus on the pro-inflammatory and pro-angiogenic effects of interleukin-1β (IL-1β) on corneal wound healing. However, the effect of IL-1β on keratocyte phenotype and corneal repair, as well as the underlying mechanisms, is not clear. This study reports, for the first time, that IL-1β induces phenotype changes of keratocytes in vitro, by significantly down-regulating the gene and protein expression levels of keratocyte markers (Keratocan, Lumican, Aldh3a1 and CD34). Furthermore, it is found that the NF-κB pathway is involved in the IL-1β-induced changes of keratocyte phenotype, and that the selective IKKβ inhibitor TPCA-1, which inhibits NF-κB, can preserve keratocyte phenotype under IL-1β simulated pathological conditions in vitro. By using a murine model of corneal injury, it is shown that sustained release of TPCA-1 from degradable silk fibroin hydrogels accelerates corneal wound healing, improves corneal transparency, enhances the expression of keratocyte markers, and supports the regeneration of well-organized epithelium and stroma. These findings provide insights not only into the pathophysiological mechanisms of corneal wound healing, but also into the potential development of new treatments for patients with corneal injuries.

Application of injectable silk fibroin/graphene oxide hydrogel combined with bone marrow mesenchymal stem cells in bone tissue engineering

The stem cell composite hydrogel scaffold was constructed following its application in bone tissue engineering. For this study, different silk fibroin (SF) hydrogels were prepared by enzyme-catalyzed crosslinking of SF and graphene oxide (GO), and their physicochemical properties and biocompatibility were examined. It has been observed that with the incorporation of GO, the pore size of the scaffold became more regular, and the porosity changed negligibly and also exhibited a stable silk II structure with excellent mechanical properties. Moreover, bone marrow stromal cells (BMSCs) were encapsulated in the hydrogel compared to SF hydrogels either in the absence or in the presence of high concentrations of GO. It has been noted that the cells in the compound gel at a low concentration of GO had shown a better growth capability, proliferation and osteogenic differentiation. Therefore, such injectable BMSCs hydrogel is an excellent biomedical material, possessing potential applications in the field of bone tissue engineering.

Tannic acid-reinforced methacrylated chitosan/methacrylated silk fibroin hydrogels with multifunctionality for accelerating wound healing

Natural polymeric hydrogel featuring multifunctional properties is more attractive as wound dressing. Herein, Tannic acid (TA)-reinforced methacrylated chitosan (CSMA)/methacrylated silk fibroin (SFMA) hydrogels were fabricated by two-step method of photopolymerization and TA solution incubating treatment. The TA in hydrogels not only served as second crosslinker improving the mechanical performance of up to a 5-fold increase (5 % TA treatment) than the pristine one, but also as functional molecule that endowed the hydrogels with enhanced adhesiveness and antioxidative properties. Besides, the introduction of TA into hydrogels further improved the antimicrobial activities against both Escherichia coli (E. coli) and Staphylococcus Aureus (S. aureus), as well as the cytocompatibility on fibroblasts. Moreover, it was demonstrated that the TA-treated CSMA/SFMA hydrogels could significantly promote wound healing in a full-thickness skin defect model. Collectively, these results showed that TA-reinforced CSMA/SFMA hydrogels could be a promising candidate as wound dressing.

Effect of gelatin on dimensional stability of silk fibroin hydrogel structures fabricated by digital light processing 3D printing

This work describes development of a silk fibroin (SF)-based ink material for digital light processing 3D printing. Riboflavin initiates the crosslinking of SF molecules under irradiation with visible light to form an SF hydrogel through dityrosine bonding. Rapid formation of a β-sheet structure induces shrinkage of SF hydrogels in phosphate-buffered saline solution at 37°C. Such shrinkage of SF hydrogel is effectively prevented by addition of gelatin to SF ink. As the ratio of gelatin in the composite ink is increased, the density of β-sheet structures is reduced, and structural deformation of the 3D SF/gelatin hydrogel is inhibited. Printability is critical in fabrication of 3D SF/gelatin composite hydrogel structures. For evaluation of printing performance, the actual width of the hydrogel formed by 3D printing is compared with the designed width. The 3D printing of SF is performed by irradiating a series of features with different shapes or sizes in a vertical manner.

Beta-sheet content significantly correlates with the biodegradation time of silk fibroin hydrogels showing a wide range of compressive modulus

Elasticity and biodegradability are required for hydrogel scaffolds in soft tissue engineering and vary among target tissues. To identify the unique effects of these properties on tissue regeneration, a factor that alters only one of these properties independent of the other is needed. Here, we evaluated various properties of 15 types of silk fibroin (SF) physical hydrogels with different SF concentrations and molecular weights, and found that the compressive modulus and biodegradability did not correlate with each other significantly. This suggested the possibility of a factor that affected only one of the properties. SF concentration and water content of the hydrogels changed both the compressive modulus and biodegradability simultaneously. In contrast, the β-sheet content in SF hydrogels correlated only with the biodegradation time significantly, suggesting that the β-sheet content may be used to study the effect of biodegradation time on tissue regeneration. In addition, an SF hydrogel was modified with a peptide derived from the β-sheet forming GAGAGS/Y repeated region in SF heavy-chain, resulting in no significant difference in the compressive modulus but a significantly delayed biodegradation time. These results suggest the potential for SF hydrogels as a group of materials with made-to-order elasticity and biodegradability for soft tissue engineering applications.

High biocompatible AuNCs-silk fibroin hydrogel system for visual detection of H2O2

Hydrogen peroxide (H2O2) quantification in biomedicine is an important indicator for different biological tissues. In this study, we developed a visual detection for H2O2 sensing based on gold nanozyme-silk fibroin (AuNCs-SF) hydrogel hybrid system with good compatibility between silk fibroin hydrogel and BSA-AuNCs. The AuNCs-SF hydrogel exhibited a fast response to H2O2 directly by fluorescence change and showed high sensitivity (limit of detection of 0.072 mM) and well stability. Moreover, the hydrogel possessed high selectivity for H2O2 in FBS solution and excellent biocompatibility to L929 cells (mouse fibroblasts). Therefore, the AuNCs-SF hydrogel hybrid system has a great potential in the fields of clinical diagnosis for visual detection of H2O2.

Body-Integrated, Enzyme-Triggered Degradable, Silk-Based Mechanical Sensors for Customized Health/Fitness Monitoring and In Situ Treatment.

Mechanical signals such as pressure and strain reflect important psychological and physiological states of the human body. Body‐integrated sensors, including skin‐mounted and surgically implanted ones, allow personalized health monitoring for the general population as well as patients. However, the development of such measuring devices has been hindered by the strict requirements for human‐biocompatible materials and the need for high performance sensors; most existing devices or sensors do not meet all the desired specifications. Here, a set of flexible, stretchable, wearable, implantable, and degradable mechanical sensors is reported with excellent mechanical robustness and compliance, outstanding biocompatibility, remotely‐triggered degradation, and excellent sensing performance, using a conductive silk fibroin hydrogel (CSFH). They can detect multiple mechanical signals such as pressure, strain, and bending angles. Moreover, combined with a drug‐loaded silk‐based microneedle array, sensor‐equipped devices are shown to be effective for real‐time monitoring and in situ treatment of epilepsy in a rodent model. These sensors offer potential applications in custom health monitoring wearables, and in situ treatment of chronic clinical disorders.

Silk fibroin hydrogel promote burn wound healing through regulating TLN1 expression and affecting cell adhesion and migration.

Skin injury is a kind of common tissue damage in daily life and war. Silk fibroin (SF) is becoming an engineered material for skin wound repair due to its superior unique physical and chemical properties. The present study aimed to illustrate mechanism of SF hydrogel promoting skin repair in the second degree burn mice. Heat shock models were established. In vitro, cells were culture for 50 min at 44 °C water bath; while in vivo, the skin of anesthetic mice were treat with soldering iron at 90 °C. Then, they divided into silk fibroin gel group, purilon gel group and control (blank) group. The cellular activity of proliferation and apoptosis was detected by Kit-8, flow cytometry and HE-staining, and the migration and adhesion were detected by scratch test. qRT-PCR and WB were employed to detected adhesion and migration related genes and proteins expression. TLN1 siRNA and overexpression technologies were also employed to illustrate the potential mechanism of SF effects. Compared with the purilon gel group and control group, SF hydrogel could enhance cell proliferation, migration and adhesion and increase the expression of adhesion and migration related proteins (P < 0.05), which promote burn wound healing. Through the inhibition, overexpression and rescue experiments of Talin1, we proved that silk fibroin hydrogel promote burn wound healing through regulating TLN1 expression and affecting cell adhesion and migration.

Injectable Silk Fibroin-Based Hydrogel for Sustained Insulin Delivery in Diabetic Rats.

Diabetes is a chronic disease affecting over 400 million people worldwide. Inadequate production of insulin due to loss of beta cells or insulin resistance within the body imbalances the glucose homeostasis, resulting in an abrupt increase of blood glucose level. The conventional and last resort of treatment involves repeated subcutaneous insulin injections to maintain the physiological glucose homeostasis. However, continuous and multiple subcutaneous injections are associated with poor patient compliance and local amyloidosis of insulin, which can be overcome with controlled and sustained insulin delivery. In this context, we have designed and formulated an injectable silk fibroin hydrogel (iSFH) to realize sustained insulin delivery over a prolonged period under diabetic conditions. The specific composition of glycol additives (ethylene glycol and triethylene glycol) allowed the silk fibroin protein to form an injectable hydrogel within 50 min. The detailed characterization of iSFH by a field-emission scanning electron microscope displayed the desired mesoporous structures, which are appropriate for drug (insulin) encapsulation in its active form. Interestingly, the subcutaneous injection of iSFH-encapsulated insulin (insulin-iSFH) in diabetic T1DM Wistar rats showed controlled release of insulin and restored physiological glucose homeostasis up to 4 days. The biocompatible and biodegradable nature of iSFH makes it a potential drug delivery system for active storage, and controlled and sustained delivery of insulin in diabetic conditions to maintain the physiological glucose level.

Enzyme-Mediated Conjugation of Peptides to Silk Fibroin for Facile Hydrogel Functionalization.

Enzymatic crosslinking of tyrosine is a simple and modular method for adding functional peptides to silk fibroin (SF) hydrogels. Silk fibroin is a naturally derived polymer notable for its robust mechanical properties, biological compatibility, and versatility. Hydrogels fabricated from SF are elastic, optically clear, and have tunable moduli, however, they do not contain native biological epitopes for cell interactions. In this work we demonstrate the attachment of peptides to SF hydrogels through crosslinking of tyrosine with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The goal was to understand the utility of this approach and to study how the addition of peptides affects the SF material properties. SF hydrogels conjugated to model peptides with different molecular weights and hydrophobic properties were studied by liquid chromatography/tandem mass spectroscopy (LC-MS/MS) (bond formation), fluorescent imaging (spatial distribution), Fourier transform infrared spectroscopy (FTIR) (protein secondary structure), and rheology (gelation time, modulus). As a proof of concept using a biologically relevant peptide, a peptide containing the cell binding domain Arg-Gly-Asp (RGD) was conjugated to SF, and the density and morphology of primary human fibroblasts were assessed. This work demonstrates a facile method for adding peptides to silk fibroin that can be adopted for a variety of biomaterials applications.

Exploring the Gelation Mechanisms and Cytocompatibility of Gold (III)-Mediated Regenerated and Thiolated Silk Fibroin Hydrogels.

Accelerating the gelation of silk fibroin (SF) solution from several days or weeks to minutes or few hours is critical for several applications (e.g., cell encapsulation, bio-ink for 3D printing, and injectable controlled release). In this study, the rapid gelation of SF induced by a gold salt (Au3+) as well as the cytocompatibility of Au3+-mediated SF hydrogels are reported. The gelation behaviors and mechanisms of regenerated SF and thiolated SF (tSF) were compared. Hydrogels can be obtained immediately after mixing or within three days depending on the types of silk proteins used and amount of Au3+. Au3+-mediated SF and tSF hydrogels showed different color appearances. The color of Au-SF hydrogels was purple-red, whereas the Au-tSF hydrogels maintained their initial solution color, indicating different gelation mechanisms. The reduction of Au3+ by amino groups and further reduction to Au by tyrosine present in SF, resulting in a dityrosine bonding and Au nanoparticles (NPs) production, are proposed as underlying mechanisms of Au-SF gel formation. Thiol groups of the tSF reduced Au3+ to Au+ and formed a disulfide bond, before a formation of Au+-S bonds. Protons generated during the reactions between Au3+ and SF or tSF led to a decrease of the local pH, which affected the chain aggregation of the SF, and induced the conformational transition of SF protein to beta sheet. The cytocompatibility of the Au-SF and tSF hydrogels was demonstrated by culturing with a L929 cell line, indicating that the developed hydrogels can be promising 3D matrices for different biomedical applications.

Addition of Platelet-Rich Plasma to Silk Fibroin Hydrogel Bioprinting for Cartilage Regeneration.

The recent advent of 3D bioprinting of biopolymers provides a novel method for fabrication of tissue-engineered scaffolds and also offers a potentially promising avenue in cartilage regeneration. Silk fibroin (SF) is one of the most popular biopolymers used for 3D bioprinting, but further application of SF is hindered by its limited biological activities. Incorporation of growth factors (GFs) has been identified as a solution to improve biological function. Platelet-rich plasma (PRP) is an autologous resource of GFs, which has been widely used in clinic. In this study, we have developed SF-based bioinks incorporated with different concentrations of PRP (12.5%, 25%, and 50%; vol/vol). Release kinetic studies show that SF-PRP bioinks could achieve controlled release of GFs. Subsequently, SF-PRP bioinks were successfully fabricated into scaffolds by bioprinting. Our results revealed that SF-PRP scaffolds possessed proper internal pore structure, good biomechanical properties, and a suitable degradation rate for cartilage regeneration. Live/dead staining showed that 3D, printed SF-PRP scaffolds were biocompatible. Moreover, in vitro studies revealed that tissue-engineered cartilage from the SF-PRP group exhibited improved qualities compared with the pure SF controls, according to histological and immunohistochemical findings. Biochemical evaluations confirmed that SF-PRP (50% PRP, v/v) scaffolds allowed the largest increases in collagen and glycosaminoglycan concentrations, when compared with the pure SF group. These findings suggest that 3D, printed SF-PRP scaffolds could be potential candidates for cartilage tissue engineering. Impact statement Three-dimensional bioprinting of silk fibroin (SF) hydrogel as bioinks is a promising strategy for cartilage tissue engineering, but it lacks biological activities, which favors proliferation of seeded cells and secretion of the extracellular matrix. In this study, we have successfully added platelet-rich plasma (PRP) into SF-based bioinks as an autologous source of growth factors. The 3D, printed SF-PRP scaffold showed an enhanced biological property, thus aiding in potential future development of novel cartilage tissue engineering applications.