Silk Hydrogels Research Articles

In vitro studies on space-conforming self-assembling silk hydrogels as a mesenchymal stem cell-support matrix suitable for minimally invasive brain application

Advanced cell therapies require robust delivery materials and silk is a promising contender with a long clinical track record. Our aim was to optimise self-assembling silk hydrogels as a mesenchymal stem cell (MSC)-support matrix that would allow future minimally invasive brain application. We used sonication energy to programme the transition of silk (1–5% w/v) secondary structure from a random coil to a stable β-sheet configuration. This allowed fine tuning of self-assembling silk hydrogels to achieve space conformity in the absence of any silk hydrogel swelling and to support uniform cell distribution as well as cell viability. Embedded cells underwent significant proliferation over 14 days in vitro, with the best proliferation achieved with 2% w/v hydrogels. Embedded MSCs showed significantly better viability in vitro after injection through a 30G needle when the gels were in the pre-gelled versus post-gelled state. Silk hydrogels (4% w/v) with physical characteristics matching brain tissue were visualised in preliminary in vivo experiments to exhibit good space conformity in an ischemic cavity (intraluminal thread middle cerebral artery occlusion model) in adult male Sprague-Dawley rats (n = 3). This study informs on optimal MSC-hydrogel matrix conditions for minimally invasive application as a platform for future experiments targeting brain repair.

Novel silk fibroin nanoparticles incorporated silk fibroin hydrogel for inhibition of cancer stem cells and tumor growth.

BackgroundA multi-drug delivery platform is needed as the intra-tumoral heterogeneity of cancer leads to different drug susceptibility. Cancer stem cells (CSCs), a small population of tumor cells responsible for tumor seeding and recurrence, are considered chemotherapy-resistant and have been reported to be sensitive to salinomycin (Sal) instead of paclitaxel (Ptx). Here we report a novel silk fibroin (SF) hydrogel-loading Sal and Ptx by incorporating drug-loaded silk fibroin nanoparticles (SF-NPs) to simultaneously kill CSCs and non-CSCs.MethodsUsing the method we have previously reported to prepare Ptx-loaded SF-NPs (Ptx-SF-NPs), Sal-loaded SF-NPs (Sal-SF-NPs) were fabricated under mild and non-toxic conditions. The drug-loaded SF-NPs were dispersed in the ultrasound processed SF solution prior to gelation.ResultsThe resulting SF hydrogel (Sal-Ptx-NP-Gel) retained its injectable properties, exhibited bio-degradability and demonstrated homogeneous drug distribution compared to the non-NP incorporated hydrogel. Sal-Ptx-NP-Gel showed superior inhibition of tumor growth compared to single drug-loaded hydrogel and systemic dual drug administration in the murine hepatic carcinoma H22 subcutaneous tumor model. Sal-Ptx-NP-Gel also significantly reduced CD44+CD133+ tumor cells and demonstrated the least tumor formation in the in vivo tumor seeding experiment, indicating superior inhibition of cancer stem cells.ConclusionThese results suggest that SF-NPs incorporated SF hydrogel is a promising drug delivery platform, and Sal-Ptx-NP-Gel could be a novel and powerful locoregional tumor treatment regimen in the future.

Characterization of silk hydrogel formed with hydrolyzed silk fibroin-methacrylate via photopolymerization

Silk fibroin (SF) hydrogel has been highlighted for its unique physical property as well as biocompatibility. SF hydrogel is easily formed from aqueous solution by physical crosslinking via β-sheet structure formation. However, such a physically formed SF hydrogel is mechanically poor with low reproducibility. Alternatively, SF hydrogel formation can be achieved by chemical cross-linking, which is able to precisely control the network density with high reproducibility. Nevertheless, chemical cross-linking process is very restricted for SF since it is very difficult to maintain homogeneous reaction condition in SF solution. To achieve controlled crosslinking, we synthesized SF-methacrylate (SFMA) with hydrolyzed SF and fabricated SF hydrogel via photo-crosslinking in the presence of photoinitiator. SFMA powder synthesized with 25 kDa SF showed good water-solubility at various degrees of methacrylation as well as quick gelation time (<60 s). In addition, the photo-crosslinked SF hydrogel showed a wide property control range with higher elasticity compared to physically crosslinked SF hydrogel.

In vivo degradation and neovascularization of silk fibroin implants monitored by multiple modes ultrasound for surgical applications

BackgroundIn this paper we aimed to investigate the neovascularization and biodegradation of the silk fibroin in vivo using multiple modes ultrasound, including two-dimensional, three-dimensional and contrast-enhanced ultrasound by quantifying the echo intensity, volume and contrast enhancement of the silk fibroin implants.MethodA total of 56 male Wistar rats were randomly divided into two groups and 4%(w/v) silk hydrogels were injected subcutaneously at hind limb or upper back of the rats respectively to compare the biodegradation rate in different sites of the body. The implants were observed at day 0, 4, 8, 12, 16, 18, 20 with multiple modes ultrasound.ResultsThe echo intensity of silk fibroin implants increased and the volume decreased gradually, and complete degradation was confirmed 18 and 20 days after subcutaneous implantation at the upper back and at the hind limb respectively. This demonstrated that the silk fibroin embedded in the upper back degraded slightly faster than that in the hind limb. Additionally, the neovascularization revealed by the contrast enhancement values of CEUS showed that there was a relatively low enhancement (< 5 dB) during day 4 to day 16, followed by moderate enhancement at day 18 (5–20 dB), and a significant enhancement at day 20 (> 40 dB).ConclusionThis study suggests that multiple modes ultrasound imaging could be an ideal method to evaluate the degradation and neovascularization of biomaterial implants in vivo for surgical applications.

Microfluidics: Tuning Enzymatically Crosslinked Silk Fibroin Hydrogel Properties for the Development of a Colorectal Cancer Extravasation 3D Model on a Chip (Global Challenges 5‐6/2018)

In order to win the battle against cancer, further advances are needed to unveil identification of cancer-causing agents in in vitro and in vivo animal models, as well as for the development of personalized therapies and drug screening. They provide insightful knowledge on the mechanisms of tumor growth and metastasis. Therefore, the transition from 2D to 3D models, comprising the integration of tissue engineering (TE) strategies with microfluidic technologies, have sparked a breakthrough in the design of in vitro microfluidic culture models. Microfluidic devices are now one of the most promising tools to mimic in-vivo-like conditions, either in normal or disease scenarios such as tumorigenesis or pathogenesis. They can provide useful model systems to investigate complex phenomena under combination of multiple controllable biochemical and biophysical microenvironments when coupled with high resolution real-time imaging. The commercially available microfluidic chip used in this study enables precise visualization of cancer cell migration within a 3D matrix in response to microenvironmental cues, shedding light on the importance of biophysical properties in cell behavior. The developed model employs enzymatically crosslinked silk fibroin hydrogels to access cancer cell migration in response to hVCAM-1, a molecule that has a major role in the extravasation process. This is precisely what is being observed in the zoom-in image: red-stained colorectal cancer cells migrating from the top of the channel towards the inside of the extracellular matrix-like silk hydrogel. Further details can be found in article number 1700100 by Joaquim M. Oliveira and co-workers.

Reverse-engineered silk hydrogels for cell and drug delivery.

Silk is an important biopolymer for (bio)medical applications because of its unique and highly versatile structure and its robust clinical track record in human medicine. Silk can be processed into many material formats, including physically and chemically cross-linked hydrogels that have almost limitless applications ranging from tissue engineering to biomedical imaging and sensing. This concise review provides a detailed background of silk hydrogels, including silk structure-function relationships, biocompatibility and biodegradation, and it explores recent developments in silk hydrogel utilization, with specific reference to drug and cell delivery. We address common pitfalls and misconceptions while identifying emerging opportunities, including 3D printing.

Recombinant Spider Silk Hydrogels for Sustained Release of Biologicals.

Therapeutic biologics (i.e., proteins) have been widely recognized for the treatment, prevention, and cure of a variety of human diseases and syndromes. However, design of novel protein-delivery systems to achieve a nontoxic, constant, and efficient delivery with minimal doses of therapeutic biologics is still challenging. Here, recombinant spider silk-based materials are employed as a delivery system for the administration of therapeutic biologicals. Hydrogels made of the recombinant spider silk protein eADF4(C16) were used to encapsulate the model biologicals BSA, HRP, and LYS by direct loading or through diffusion, and their release was studied. Release of model biologicals from eADF4(C16) hydrogels is in part dependent on the electrostatic interaction between the biological and the recombinant spider silk protein variant used. In addition, tailoring the pore sizes of eADF4(C16) hydrogels strongly influenced the release kinetics. In a second approach, a particles-in-hydrogel system was used, showing a prolonged release in comparison with that of plain hydrogels (from days to week). The particle-enforced spider silk hydrogels are injectable and can be 3D printed. These initial studies indicate the potential of recombinant spider silk proteins to design novel injectable hydrogels that are suitable for delivering therapeutic biologics.

Coding cell micropatterns through peptide inkjet printing for arbitrary biomineralized architectures.

Well-designed micropatterns present in native tissues and organs involve changes in extracellular matrix compositions, cell types and mechanical properties to reflect complex biological functions. However, the design and fabrication of these micropatterns in vitro to meet task-specific biomedical applications remains a challenge. A de novo design strategy to code and synthesize functional micropatterns is presented to engineer cell alignment through the integration of aqueous-peptide inkjet printing and site-specific biomineralization. The inkjet printing provides direct writing of macroscopic biosilica selective peptide-R5 patterns with micrometer-scale resolution on the surface of a biopolymer (silk) hydrogel. This is combined with in situ biomineralization of the R5 peptide for site-specific growth of silica nanoparticles on the micropatterns, avoiding the use of harsh chemicals or complex processing. The functional micropatterned systems are used to align human mesenchymal stem cells and bovine serum albumin. This combination of peptide printing and site-specific biomineralization provides a new route for developing cost-effective micropatterns, with implications for broader materials designs. Coding cell micropatterns through peptide inkjet printing for arbitrary biomineralized architectures is demonstrated here. The functional micropatterned systems are used to align human mesenchymal stem cells and bovine serum albumin in vitro, avoiding the use of harsh chemicals or complex processing, while providing potential applications in developing cost-effective micropatterns to meet task-specific biomedical applications.

Bioactive Silk Hydrogels with Tunable Mechanical Properties.

Developing bioactive hydrogels with potential to guide the differentiation behavior of stem cells has become increasingly important in the biomaterials field. Here, silk hydrogels with tunable mechanical properties were developed by introducing inert silk fibroin nanofibers (SNF) within an enzyme crosslinked system of regenerated silk fibroin (RSF). After the crosslinking reaction of RSF, the inert SNF was embedded into the RSF hydrogel matrix, resulting in improved mechanical properties. Tunable stiffness in the range of 9-60 KPa was achieved by adjusting the amount of the added NSF, significantly higher than SNF-free hydrogels formed under same conditions (about 1 KPa). In addition, the proliferation of rat bone marrow derived mesenchymal stem cells cultured on the composite hydrogels and differentiated into endothelial cells, myoblast and osteoblast cells was improved, putatively due to the control of stiffness of the hydrogels. Bioactive and tunable silk-based hydrogels were prepared via a composite SNF and crosslinked RSF system, providing a new strategy to design silk biomaterials with tunable mechanical and biological performance.

Antibacterial PET-Silk fabric containing Salvia officinalis extract

ABSTRACTIn this study, novel PET-Silk fabric is introduced via finishing PET fabric with silk hydrogel and Salvia officinalis extract. The silk hydrogel was prepared from waste silk; morphology and chemical structure were evaluated by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy methods. The results indicated the 3D porous network and crystalline structure of hydrogel. The polyester fabric was finished with prepared hydrogel. Contact angle and water uptake of modified fabric were assessed. S. officinalis extract was used as an antibacterial agent. Results of antibacterial tests confirmed a remarkable antibacterial effect of finished fabrics with the extract.

Tuning the structure and performance of silk biomaterials by combining mulberry and non-mulberry silk fibroin

Despite considerable efforts toward the biomedical applications of silk fibroin (SF), detailed control of structure and performance in silk biomaterials remains a major challenge. Silks are classified as mulberry and non-mulberry, which are produced by domesticated Bombyx mori and wild silkworm species, respectively. To achieve silk biomaterials with tunable performance, Bombyx mori silk fibroin (BSF) and non-mulberry Antheraea pernyi silk fibroin (ASF) were blended. These systems exploit the beneficial material and biological properties of both silk proteins. The sol-gel transition of ASF was much more rapid than that of BSF. Consequently, the gelation rate of SF solution could be controlled ranging from tens of minutes to days by changing the ASF ratio, providing useful options for the fabrication of silk hydrogels. Raising BSF ratio in the blend materials significantly increased mechanical performance, overcoming the inherent lack of mechanical performances of regenerated ASF materials. Conversely, increasing ASF ratio significantly enhanced degradability of the blend films, providing a wider range of regulation for the degradation rate of silk biomaterials. As a result, this simple blending strategy enables the process to be more controllable and flexible, controlling the performance of silk protein becomes feasible, and this would greatly expand the applications of silk as a biomedical material.

Recombinant spider silk-based bioinks

Bioinks, 3D cell culture systems which can be printed, are still in the early development stages. Currently, extensive research is going into designing printers to be more accommodating to bioinks, designing scaffolds with stiff materials as support structures for the often soft bioinks, and modifying the bioinks themselves. Recombinant spider silk proteins, a potential biomaterial component for bioinks, have high biocompatibility, can be processed into several morphologies and can be modified with cell adhesion motifs to enhance their bioactivity. In this work, thermally gelled hydrogels made from recombinant spider silk protein encapsulating mouse fibroblast cell line BALB/3T3 were prepared and characterized. The bioinks were evaluated for performance in vitro both before and after printing, and it was observed that unprinted bioinks provided a good platform for cell spreading and proliferation, while proliferation in printed scaffolds was prohibited. To improve the properties of the printed hydrogels, gelatin was given as an additive and thereby served indirectly as a plasticizer, improving the resolution of printed strands. Taken together, recombinant spider silk proteins and hydrogels made thereof show good potential as a bioink, warranting further development.

An Insight on Silk Protein Sericin: From Processing to Biomedical Application.

Silks are naturally occurring polymers that have been used clinically as sutures for hundreds of years. It's so for obtained from insects or worms, silk consists of a filament core protein, termed fibroin, and a glue-like coating made up of sericin proteins. An important component of silk has an extended history of being discarded as a waste in the course of silk processing. The cost of sericin for tissue engineering is underestimated and its capability in using as regenerative remedy has simply began to be explored. Its variable amino acid composition and various functional groups confer upon it attractive bioactive proteins, which are particularly interesting for biomedical programs. Because of its antioxidant properties, moisturizing ability, and mitogenic effect on mammalian cells, sericin is beneficial in cell regeneration and tissue engineering. Research shows that keratinocytes and fibroblasts have brought about the improvement of sericin-primarily based biomaterials for skin tissue repair, in particular as wound dressings. Moreover, sericin may be used for bone tissue engineering due to its ability to set off nucleation of bone-like hydroxyapatite. Stable silk sericin biomaterials, as films, sponges, and hydrogels, are obtained by means of cross-linking, ethanol precipitation, or mixing with different polymers. Now a day, sericin may also be used for delivery of drugs due to its chemical reactivity and pH-responsiveness which facilitate the fabrication of nano and microparticles, hydrogels, and conjugated molecules, enhancing the bioactivity of drugs. In this review, we outlined the current headways from extraction of sericin till its physical properties and biomedical applications.

High water content silk protein-based hydrogels with tunable elasticity fabricated via a Ru(II) mediated photochemical cross-linking method

Silk is very promising in the field of biomaterials as a natural biomacromolecule. Silk protein can be made into various forms of materials, including hydrogels. However, silk protein-based hydrogels have not attracted much attention due to its weak mechanical properties. Here, we report high water content silk protein-based hydrogels with tunable elasticity which were fabricated through Ru(II) mediated photochemically cross-linking tyrosine residues in regenerated silk protein. The regenerated silk protein was characterized by Fourier transform infrared spectroscopy (FTIR). The gelation kinetics of the silk protein was studied by rheology measurements. The compressive mechanical properties of the silk protein-based hydrogels was investigated using compressive tests and dynamic mechanical analysis (DMA). Compressive modulus of the hydrogels reached 349±64 MPa at 15 % strain. The fabricated silk protein-based hydrogels were also characterized by Scanning electron microscopy (SEM), revealing an interconnected porous network structure, typical of hydrogels, with an average pore size of approximately 130 μm. Finally, biocompatibility of the silk protein-based hydrogels was demonstrated through cell culture studies using a human fibroblast cell line, HFL1. The reported silk protein-based hydrogels represent a promising candidate for biomaterial applications.

Effect of stiffness change of transplanted induced neural stem cells after spinal cord injury using silk hydrogel

Effect of stiffness change of transplanted induced neural stem cells after spinal cord injury using silk hydrogel

Dissolution and regeneration of non-mulberry Eriogyna Pyretorum silk fibroin

Protein-based materials have been actively pursued as biomaterials because of their nontoxicity, biocompatibility and biodegradability. In this work, we demonstrated the potential of Eriogyna pyretorum silk fibroin (ESF), a non-mulberry silk protein, as biomaterials. The degummed ESF fibers could be dissolved completely by Ca(NO3)2/H2O/C2H5OH solution to produce regenerated ESF. The solubility was strongly dependent on the addition of C2H5OH, heating temperature and dissolving time. α-helix and random coil are main molecular conformation in aqueous ESF solution. The sol–gel transition behavior of regenerated ESF was also studied, indicating that the conformational transition of regenerated ESF from random coil/α-helix to β-sheet during gelation. Especially, ESF showed more rapid gelation than mulberry silk fibroin (BSF). Consequently, the gelation rate of BSF could be controlled ranging from tens of minutes to days by changing the ESF ratio, providing useful options for the fabrication of silk hydrogels. Water-stable regenerated ESF film could be achieved by using aqueous ethanol to induce structural transition. Tensile tests showed that the ESF films have a dry strength of approximate 31.0 MPa and a wet strength of approximate 3.3 MPa. This study provides new opportunities as an alternative natural protein material for biomedical applications.

Temperature buffering capacity of silk hydrogel: A useful packaging material

Abstract: The n-octadecane incorporated silk hydrogel is developed by the gelation of an o/w emulsion made of n-octadecane and the aqueous silk solution acting as the oil and water phases, respectively. SEM images show that n-octadecane grains can be clearly observed in the pores of the lyophilized hydrogel, which indicates the successful embedment of n-octadecane in the composite hydrogel. Furthermore, the composite hydrogel exhibits good water absorption capability and thermal regulation capacity. Therefore the silk composite hydrogel can be a promising candidate as the potential packaging structures for temperature sensitive materials.

Molecular and macro-scale analysis of enzyme-crosslinked silk hydrogels for rational biomaterial design.

Hydrogels from naturally-derived silk polymers offer versitile opportunities in the biomedical field, however, their design has largely been an empirical process. We present a fundamental study of the crosslink density, storage modulus, and diffusion behavior of enzymatically-crosslinked silk hydrogels to better inform scaffold design. These studies revealed unexpected non-linear trends in the crosslink density and diffusivity of silk hydrogels with respect to protein concentration and crosslink reagent concentration. This work demonstrates the tunable diffusivity and crosslinking in silk fibroin hydrogels, and enables the rational design of biomaterials. Further, the characterization methods presented have applications for other materials with dityrosine crosslinks, which are found in nature as post-translational modificaitons, as well as in engineered matrices such as tyramine-substituted hyaluronic acid and recombinant resilin.

Characterization of Hydrogels Made of a Novel Spider Silk Protein eMaSp1s and Evaluation for 3D Printing.

Recombinantly produced spider silk proteins have high potential for bioengineering and various biomedical applications because of their biocompatibility, biodegradability, and low immunogenicity. Here, the recently described small spider silk protein eMaSp1s is assembled into hydrogels, which can be 3D printed into scaffolds. Further, blending with a recombinantly produced MaSp2 derivative eADF4(C16) alters the mechanical properties of the resulting hydrogels. Different spider silk hydrogels also show a distinct recovery after a high shear stress deformation, exhibiting the tunability of their features for selected applications.

Silk-hydrogel Lenses for Light-emitting Diodes

Today the high demand for electronics leads to massive production of waste, thus green materials based electronic devices are becoming more important for environmental protection and sustainability. The biomaterial based hydrogels are widely used in tissue engineering, but their uses in photonics are limited. In this study, silk fibroin protein in hydrogel form is explored as a bio-friendly alternative to conventional polymers for lens applications in light-emitting diodes. The concentration of silk fibroin protein and crosslinking agent had direct effects on optical properties of silk hydrogel. The spatial radiation intensity distribution was controlled via dome- and crater-type silk-hydrogel lenses. The hydrogel lens showed a light extraction efficiency over 0.95 on a warm white LED. The stability of silk hydrogel lens is enhanced approximately three-folds by using a biocompatible/biodegradable poly(ester-urethane) coating and more than three orders of magnitude by using an edible paraffin wax coating. Therefore, biomaterial lenses show promise for green optoelectronic applications.