Structure of silk I (Bombyx mori silk fibroin before spinning) in the dry and hydrated states studied using 13C solid-state NMR spectroscopy.

Nowadays, much attention has been paid to Bombyx mori silk fibroin (SF) by many researchers because of excellent physical properties and biocompatibility. These superior properties originate from the structure of SF and therefore, the structural analysis is a key to clarify the superiority. Here we concentrated on silk I structure (SF structure before spinning). We showed that silk I* (the structure of (GAGAGS)n which is a main part of SF) is a repeated type II ß-turn, neither α-helix nor random coil, from the conformation-dependent 13C NMR chemical shift data. This conclusion is different from that obtained using IR by many researchers. Next, the formation of silk I* structure was investigated at molecular level using 13C solid-state NMR spectroscopy. Three kinds of 13C INEPT, CP/MAS and DD/MAS NMR spectra were observed for SF, [3-13C]Ser- and [3-13C]Tyr-SF, the crystalline fraction obtained by chymotrypsin treatment of SF and their model peptide with silk I structures in the dry and hydrated states. Especially, the presence of the sequences containing Tyr, (((GX)m1GY)m2 where X = A or V) with random coil conformations adjacent to (GAGAGS)n is an essence to get water-soluble SF and the formation of silk I* structure of (GAGAGS)n.

Fabrication of silk fibroin film enhanced by acid hydrolyzed silk fibroin nanowhiskers to improve bacterial inhibition and biocompatibility efficacy.

In this study, silk fibroin nanowhiskers (SNWs) were extracted from natural silk fiber by sulfuric acid hydrolysis with the assistance of ultrasonic wave treatment. The obtained SNWs were mixed with regenerated silk fibroin (RSF) solution to fabricate the SNWs/RSF films. The fabricating SNWs were systematically characterized by using SEM, FTIR, and the SNWs/RSF films were observed by digital camera, PM, etc. The results show that the monodisperse SNWs are evenly distributed in the RSF film. The presence of SNWs in RSF film significantly improves the performances of the film, including the swelling ability, mechanical properties, hydrophilicity, antibacterial efficacy, cytocompatibility. Meanwhile, the SNWs/RSF film can endorse the wound healing efficiency in vivo mice wound site. The proposed techniques for extracting SNWs and fabricating silk fibroin composite film may provide a valuable method for creating an ideal silk-based material for biomedical applications.

Silk fibroin nanocomposites as tissue engineering scaffolds - A systematic review.

Silk fibroin is a protein with intrinsic characteristics that make it a good candidate as a scaffold for tissue engineering. Recent works have enhanced its benefits by adding inorganic phases that interact with silk fibroin in different ways. A systematic review was performed in four databases to study the physicochemical and biological performance of silk fibroin nanocomposites. In the last decade, only 51 articles contained either in vitro cell culture models or in vivo tests. The analysis of such works resulted in their classification into the following scaffold types: particles, mats and textiles, films, hydrogels, sponge-like structures, and mixed conformations. From the physicochemical perspective, the inorganic phase imbued in silk fibroin nanocomposites resulted in better stability and mechanical performance. This review revealed that the inorganic phase may be associated with specific biological responses, such as neovascularisation, cell differentiation, cell proliferation, and antimicrobial and immunomodulatory activity. The study of nanocomposites as tissue engineering scaffolds is a highly active area mostly focused on bone and cartilage regeneration with promising results. Nonetheless, there are still many challenges related to their application in other tissues, a better understanding of the interaction between the inorganic and organic phases, and the associated biological response.

Molecularly Imprinted Silk Fibroin Nanoparticles.

Nanosized biomimetics prepared by the strategy of molecular imprinting, that is, the stamping of recognition sites by means of a template-assisted synthesis, are demonstrating potential as plastic antibodies in medicine, proving effective for cell imaging and targeted therapies. Most molecularly imprinted nanoparticles (MIP-NPs) are currently made of soft matter, such as polyacrylamide and derivatives. Yet, MIP-NPs biocompatibility is crucial for their effective translation into clinical uses. Here, we propose the original idea to synthesize fully biocompatible molecularly imprinted nanoparticles starting from the natural polymer silk fibroin (MIP SF-NPs), which is nontoxic and highly biocompatible. The conditions to produce MIP SF-NPs of different sizes (dmean ∼ 50 nm; dmean ∼ 100 nm) were set using the response surface method. The stamping of a single, high affinity (KD = 57 × 10-9 M), and selective recognition site per silk fibroin nanoparticle was demonstrated, together with the confirmation of nontoxicity. Additionally, MIP SF-NPs were used to decorate silk microfibers and silk nanofibers, providing a general means to add entailed biofunctionalities to materials.

Properties of Biomimetic Artificial Spider Silk Fibers Tuned by PostSpin Bath Incubation.

Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that ≥5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased ß-sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.

Facile synthesis of 3D silk fibroin scaffolds with tunable properties for regenerative medicine.

Silk fibroin (SF) porous scaffolds provide mechanical support and biochemical signals to encourage cell attachment and modify biological performance. The available methods for the preparation of SF scaffolds are still required. The crosslinkers used are likely to impact the biocompatibility. Herein, water-insoluble SF scaffolds were prepared by physical methods. The phosphate solution promoted SF molecules aggregate from SF/heparin mixed solution. Then SF scaffolds were prepared in centrifuge tubes under different centrifugal speed. The phosphate was leached from the scaffolds, leaving porous structure. The centrifugal force produced shear-induced silk crystallinity to tune the mechanical performance like the natural silkworm gland. The relationship between performance and second structure of the scaffolds have been revealed by X-ray Diffraction (XRD) and deconvoluting Fourier Transform Infrared spectroscopy (FTIR). Due to changes in the ß-sheet content, pore structure, mechanical strength, and drug-loaded behavior was adjustable. The scaffolds performed excellent on the Activated Partial Thromboplastin Time (APTT) value, and it can keep sustainable released for 7 days. The scaffolds prepared in mild environment showed tunable stiffness, good anticoagulation, and improved cell compatibility, suggesting its potential application in regenerative medicine.

Flexible Biosensors for the Impedimetric Detection of Protein Targets Using Silk-Conductive Polymer Biocomposites.

To expand the applications of flexible biosensors in point-of-care healthcare applications beyond monitoring of biophysical parameters, it is important to devise strategies for the detection of various proteins and biomarkers. Here, we demonstrate a flexible, fully organic, biodegradable, label-free impedimetric biosensor for the critical biomarker, vascular endothelial growth factor (VEGF). This biosensor was constructed by photolithographically patterning a conducting ink consisting of a photoreactive silk sericin coupled with a conducting polymer. These functional electrodes are printed on flexible fibroin substrates that are controllably thick and can be free-standing, or conform to soft surfaces. Detection was accomplished via the antibody to VEGF which was immobilized within the conducting matrix. The results indicated that the developed flexible biosensor was highly sensitive and selective to the target protein, even in challenging biofluids such as human serum. The biosensors themselves are biocompatible and degradable. Through this work, the developed flexible biosensor based on a simple and label-free strategy can find practical applications in the monitoring of wound healing or early disease diagnosis.

LBL deposition of chitosan and silk fibroin on nanofibers for improving physical and biological performance of patches.

Pelvic floor dysfunction diseases (PFD) become more prevalent with the increase of elderly population, and complications of pelvic floor reconstructive surgery (e.g. infection and exposure of mesh) have been troubling to patients and gynecologists. In this study, the nanofibrous mats were prepared by alternately depositing chitosan (CS) and silk fibroin (SF) on Nylon6 (N6) nanofibrous mats via layer-by-layer (LBL) technique. The as-prepared mats were characterized. The results showed that CS and SF molecules were successfully assembled on the nanofibers. Additionally, after LBL modification, the hydrophilicity of the nanofibrous mats was reduced and the mechanical properties were improved. Furthermore, the antibacterial activity of the LBL-structured mats reached >95% inhibiting Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The in vitro cell co-culture experiments indicated that LBL-structured mats had smaller toxic effects and more excellent biocompatibility to L929 fibroblasts, especially the mats with 15 bilayers coated films. Hence, the LBL-structured mats are promising materials for pelvic floor reconstruction to reduce postoperative pelvic complication rates.

Method for the Destruction of Endotoxin in Synthetic Spider Silk Proteins.

Although synthetic spider silk has impressive potential as a biomaterial, endotoxin contamination of the spider silk proteins is a concern, regardless of the production method. The purpose of this research was to establish a standardized method to either remove or destroy the endotoxins present in synthetic spider silk proteins, such that the endotoxin level was consistently equal to or less than 0.25 EU/mL, the FDA limit for similar implant materials. Although dry heat is generally the preferred method for endotoxin destruction, heating the silk proteins to the necessary temperatures led to compromised mechanical properties in the resultant materials. In light of this, other endotoxin destruction methods were investigated, including caustic rinses and autoclaving. It was found that autoclaving synthetic spider silk protein dopes three times in a row consistently decreased the endotoxin level 10-20 fold, achieving levels at or below the desired level of 0.25 EU/mL. Products made from triple autoclaved silk dopes maintained mechanical properties comparable to products from untreated dopes while still maintaining low endotoxin levels. Triple autoclaving is an effective and scalable method for preparing synthetic spider silk proteins with endotoxin levels sufficiently low for use as biomaterials without compromising the mechanical properties of the materials.

Biocompatible silk/calcium silicate/sodium alginate composite scaffolds for bone tissue engineering.

Scaffolds are crucial for bone tissue engineering since their compositions and properties could significantly affect the seeded cells' behavior. In this study, we developed an interpenetrating network hydrogel by utilizing Ca2+ from calcium silicate (CS) to simultaneously crosslink silk fibroin (SF) and sodium alginate (SA). Afterwards, the hydrogels were lyophilized to obtain scaffolds and systematically evaluated by physical characterizations, in vitro cytocompatibility and alkaline phosphatase (ALP) assay. We found that CS inside the porous structure of SF/CS/SA scaffolds could remarkably enhance hydrophilicity, degradation, compression resistance, bioactivity and pH of SF/CS/SA scaffolds. Scaffolds with CS concentrations of 25% and 12% (25/CS and 12/CS) could dominantly stimulate proliferation of bone marrow stromal cells (BMSCs). Besides, BMSCs cultured with 25/CS and 12/CS scaffolds showed high ALP activity, respectively. Consequently, this study suggested SF/CS/SA scaffolds possess potential in non-loading bone tissue engineering application.

Lithium-free processing of silk fibroin.

Silk fibroin protein was purified from Bombyx mori silkworm cocoons using a novel dialysis strategy to avoid fibroin aggregation and pre-mature formation of ß-sheets. The degummed silk fibers were dissolved in Ajisawa's reagent, a mixture of CaCl2-EtOH-H2O, that is less expensive than lithium bromide. The dissolved solutions were dialyzed against either water or urea solution with a stepwise decrease in concentration. When the steps of 4 M-2 M-1 M-0 M urea (referred to as silk-TS-4210) were adopted, the purified silk fibroin had smaller aggregates (<10 nm), similar average molecular weight (225 kDa) and a lower content of ß-sheet (∼15%) compared to the sample processing methods (silk-TS-210, 10, 0) studied here. This outcome was close to the fibroin purified by the lithium bromide (silk-Li-0) method. Polyvinyl alcohol-emulsified silk microspheres generated using the purified solution had a similar size distribution and morphology when compared to lithium bromide dissolved solutions, while glycerol-blended silk films showed different mechanical properties. The silk-Li-0 generated films with the highest breaking strength (5.7 MPa ± 0.3) while the silk-TS-4210 had the highest extension at break (215.1% ± 12.5). The films prepared from silk-TS-4210 were cytocompatible to support the adhesion and proliferation of human mesenchymal stem cells, with improvements compared to the other samples likely due to the porous morphology of these films.

Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form ß-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants.

[Fundamental bases for the use of silk fibroin-based bioresorbable microvehicles as an example of skin regeneration in therapeutic practice].

AIM: To assess whether silk fibroin-based microvehicles (MVs) may be used to grow fibroblasts (FBs) and keratinocytes (KCs), key cellular components in skin regeneration after injury. SUBJECTS AND METHODS: Cryogrinding was applied to derive MVs from fibroin-based and fibroin- and 30% gelatin-containing composite matrices. To examine the structure of the matrices and MVs, confocal microscopy was used to conjugate the polymer with the dye tetramethylrhodamine isothiocyanate. Microparticle size distribution was estimated by granulometric analysis. 3T3 mouse FBs and cultured primary mouse KCs expressing green fluorescent protein (GFP) were used to study whether fibroin-based MVs might be suitable for growing the cells involved in skin regeneration. KC growth was analyzed by confocal laser scanning microscopy from cellular GFP expression. The proliferation rate of FBs and KCs was estimated by a MTT assay. RESULTS: There were two derived MV types: fibroin-based and fibroin and 30% gelatin-containing composite ones. On day 1, 3T3 mouse FBs on the fibroin-based gelatin-free MVs actively proliferated and the presence of gelatin in MVs diminished the proliferation of these cells. Fibroin-based MVs were shown to be suitable for the effective in vitro growth of KCs expressing cytokeratins 5 and 14, the major markers of KCs in the basal layer. Gelatin did not give rise to accelerated KC growth. The investigation has demonstrated that is possible to regulate FB proliferation on MVs, which is of great importance in delivering the cells into the site of injury since intensive proliferation of FBs may lead to the development of fibrosis and the formation of scar tissue. Balanced FB growth is essential to the creation of optimal conditions for KC growth in composite tissue-engineering constructions. CONCLUSION: The use of fibroin-based MVs is promising for the design of novel therapeutic materials and injectable cell therapy for different diseases.

Factors controlling the deposition of silk fibroin nanofibrils during layer-by-layer assembly.

The layer-by-layer technique has been used as a powerful method to produce multilayer thin films with tunable properties. When natural polymers are employed, complicated phenomena such as self-aggregation and fibrilogenesis can occur, making it more difficult to obtain and characterize high-quality films. The weak acid and base character of such materials provides multilayer systems that may differ from those found with synthetic polymers due to strong self-organization effects. Specifically, LbL films prepared with chitosan and silk fibroin (SF) often involve the deposition of fibroin fibrils, which can influence the assembly process, surface properties, and overall film functionality. In this case, one has the intriguing possibility of realizing multilayer thin films with aligned nanofibers. In this article, we propose a strategy to control fibroin fibril formation by adjusting the assembly partner. Aligned fibroin fibrils were formed when chitosan was used as the counterpart, whereas no fibrils were observed when poly(allylamine hydrochloride) (PAH) was used. Charge density, which is higher in PAH, apparently stabilizes SF aggregates on the nanometer scale, thereby preventing their organization into fibrils. The drying step between the deposition of each layer was also crucial for film formation, as it stabilizes the SF molecules. Preliminary cell studies with optimized multilayers indicated that cell viability of NIH-3T3 fibroblasts remained between 90 and 100% after surface seeding, showing the potential application of the films in the biomedical field, as coatings and functional surfaces.

Dragline silk: a fiber assembled with low-molecular-weight cysteine-rich proteins.

Dragline silk has been proposed to contain two main protein constituents, MaSp1 and MaSp2. However, the mechanical properties of synthetic spider silks spun from recombinant MaSp1 and MaSp2 proteins have yet to approach natural fibers, implying the natural spinning dope is missing critical factors. Here we report the discovery of novel molecular constituents within the spinning dope that are extruded into dragline silk. Protein studies of the liquid spinning dope from the major ampullate gland, coupled with the analysis of dragline silk fibers using mass spectrometry, demonstrate the presence of a new family of low-molecular-weight cysteine-rich proteins (CRPs) that colocalize with the MA fibroins. Expression of the CRP family members is linked to dragline silk production, specifically MaSp1 and MaSp2 mRNA synthesis. Biochemical data support that CRP molecules are secreted into the spinning dope and assembled into macromolecular complexes via disulfide bond linkages. Sequence analysis supports that CRP molecules share similarities to members that belong to the cystine slipknot superfamily, suggesting that these factors may have evolved to increase fiber toughness by serving as molecular hubs that dissipate large amounts of energy under stress. Collectively, our findings provide molecular details about the components of dragline silk, providing new insight that will advance materials development of synthetic spider silk for industrial applications.

Controlling the cell adhesion property of silk films by graft polymerization.

We report here a graft polymerization method to improve the cell adhesion property of Bombyx mori silk fibroin films. B. mori silk has evolved as a promising material for tissue engineering because of its biocompatibility and biodegradability. However, silk's hydrophobic character makes cell adhesion and proliferation difficult. Also, the lack of sufficient reactive amino acid residues makes biofunctionalization via chemical modification challenging. Our study describes a simple method that provides increased chemical handles for tuning of the surface chemistry of regenerated silk films (SFs), thus allowing manipulation of their bioactivity. By grafting pAAc and pHEMA via plasma etching, we have increased carboxylic acid and hydroxyl groups on silk, respectively. These modifications allowed us to tune the hydrophilicity of SFs and provide functional groups for bioconjugation. Our strategy also allowed us to develop silk-based surface coatings, where spatial control over cell adhesion can be achieved. This control over cell adhesion in a particular region of the SFs is difficult to obtain via existing methods of modifying the silk fibroin instead of the SF surface. Thus, our strategy will be a valuable addition to the toolkit of biofunctionalization for enhancing SFs' tissue engineering applications.

Synthesis and characterization of water-soluble silk peptides and recombinant silk protein containing polyalanine, the integrin binding site, and two glutamic acids at each terminal site as a possible candidate for use in bone repair materials.

The recombinant proteins [EE(A)12EETGRGDSPAAS]n (n = 5,10) were prepared as a potential scaffold material for bone repair. The construct was based on Antheraea perni silk fibroin to which cells adhere well and combined poly(alanine), the integrin binding site TGRGDSPA, and a pair of glutamic acids (E2) at both the N- and C-terminal sites to render the construct water-soluble and with the hope that it might enhance mineralization with hydroxyapatite. Initially, two peptides E2(A)nE2TGRGDSPAE2(A)nE2 (n = 6, 12) were prepared by solid state synthesis to examine the effect of size on conformation and on cell binding. The larger peptide bound osteoblasts more readily and had a higher helix content than the smaller one. Titration of the side chain COO(-) to COOH of the E2 and D units in the peptide was monitored by solution NMR. On the basis of these results, we produced the related recombinant His tagged protein [EE(A)12EETGRGDSPAAS]n (n = 5,10) by expression in Escherichia coli . The solution NMR spectra of the recombinant protein indicated that the poly(alanine) regions are helical, and one E2 unit is helical and the other is a random coil. A molecular dynamics simulation of the protein supports these conclusions from NMR. We showed that the recombinant protein, especially, [EE(A)12EETGRGDSPAAS]10 has some of the properties required for bone tissue engineering scaffold including insolubility, and evidence of enhanced cell binding through focal adhesions, and enhanced osteogenic expression of osteoblast-like cells bound to it, and has potential for use as a bone repair material.

[Study of the preparation of silk fibroin gel and its morphology as drug release matrix in vitro and in vivo].

Silk fibroin (SF)/sodium alginate (SA) hydrogels can be used as drug injection materials. Homogenate was prepared by centrifugation of the pig myocardial extracellular matrix (PMM) and its modification of SF gel material. This paper observes and compares the different components SF, SF/SA, SF/SA/PMM to illustrate the SF/SA/PMM ternary material as a drug delivery composition material. This ternary material can shorten the gel time, and can make the gel form to be maintained better. Meanwhile, it makes the internal structure of the gel looser so that the hole wall becomes thinner and more conducive to the drug release. In addition, it has good biocompatibility proved by pathological analysis, and is able to enhance the mesenchymal stem cells growth activity, which has great significance in carrying out drug control release.

Engineered spider silk protein-based composites for drug delivery.

Silk protein-based materials are promising materials for the delivery of drugs and other active ingredients, due to their processability, biocompatibility, and biodegradability. The preparation of films composed of an engineered spider silk protein (eADF4(C16)) in combination with either a polyester (polycaprolactone) or a polyurethane (pellethane), and their physical properties are described. The release profiles are affected by both the film composition and the presence of enzymes, and release can be observed over a period of several weeks. Such silk-based composites have potential as drug eluting biocompatible coatings or implantable devices.

Silk microgels formed by proteolytic enzyme activity.

The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study.