Silkworm Gut Fibres from Silk Glands of Samia cynthia ricini-Potential Use as a Scaffold in Tissue Engineering.

High-performance fibroin fibres are ideal candidates for the manufacture of scaffolds with applications in tissue engineering due to the excellent mechanical properties and optimal biocompatibility of this protein. In this work, the manufacture of high-strength fibres made from the silk glands of Samia cynthia ricini is explored. The glands were subjected to soaking in aqueous dissolutions of acetic acid and stretched to manufacture the fibres. The materials produced were widely characterized, in terms of morphology, mechanical properties, crystallinity and content of secondary structures, comparing them with those produced by the standard procedure published for Bombyx mori. In addition, mechanical properties and biocompatibility of a braided scaffold produced from these fibres was evaluated. The results obtained show that the fibres from B. mori present a higher degree of crystallinity than those from S. c. ricini, which is reflected in higher values of elastic modulus and lower values of strain at break. Moreover, a decrease in the elongation values of the fibres from S. c. ricini was observed as the concentration of acetic acid was increased during the manufacture. On the other hand, the study of the braided scaffolds showed higher values of tensile strength and strain at break in the case of S. c. ricini materials and similar values of elastic modulus, compared to those of B. mori, displaying both scaffolds optimal biocompatibility using a fibroblast cell line.

Products of Sericulture and Their Hypoglycemic Action Evaluated by Using the Silkworm, Bombyx mori (Lepidoptera: Bombycidae), as a Model.

Sericulture generates different natural products with potential medical applications. Silk peptides, worms, or even pupae are commonly employed in traditional Asian medicine with a wide variety of purposes, and some scientific work has been focused on their antidiabetic properties. This work evaluates the postprandial antihyperglycemic activity of fibroin, sericin, and powder made from either larvae or pupae of silkworms, and Bombyx mori L. (Lepidoptera: Bombycidae), employing the silkworm itself as an animal model. The results indicate a reduction in the glucose levels in hemolymph after sucrose or glucose-induced hyperglycemia when these products are included in the diet of the worms.

Effect of Yerba Mate and Silk Fibroin Nanoparticles on the Migration Properties in Ethanolic Food Simulants and Composting Disintegrability of Recycled PLA Nanocomposites.

The main objective of the present research is to study the effect of the incorporation of low amounts of silk fibroin nanoparticles (SFNs) and yerba mate nanoparticles (YMNs) on the migration phenomenon into ethanolic food simulants as well as on the disintegrability under composting conditions of mechanically recycled polylactic acid (PLA). Recycled PLA was obtained under simulated recycling conditions by melt processing virgin PLA into films and further subjecting them to an accelerated aging process, which involved photochemical, thermal, and hydrothermal aging steps followed by an intense washing step. SFNs were extracted from Bombyx mori cocoons and YMNs from yerba mate waste. Then, recycled PLA was melted, reprocessed, and reinforced with either 1%wt. of SFNs or YMNs, by melt extrusion, and further processed into films by compression molding. The obtained nanocomposites were exposed to ethanolic food simulants (ethanol 10% v/v, simulant A and ethanol 50% v/v, simulant D1) and the structural, thermal, and mechanical properties were studied before and after the exposure to the food simulants. The migration levels in both food simulants were below the overall migration limits required for food contact materials. The materials were disintegrated under simulated composting conditions at the laboratory scale level and it was observed that the nanoparticles delayed the disintegration rate of the recycled PLA matrix, but nanocomposites were fully disintegrated in less than one month.

Influence of addition of organic fillers on the properties of mechanically recycled PLA.

Poly(lactic acid) (PLA) is one of the most used biobased and biodegradable polymers. Due to their high stability, some of the newest grades of PLA are only degradable under severe industrial conditions. For these grades, mechanical recycling is a viable end-of-life option, with great environmental advantages. However, the polymer undergoes degradation during its service life and in the melt reprocessing, which leads to a decrease in properties that can compromise the recyclability of PLA. The goal of this work was to evaluate the usefulness of adding small amounts of two organic fillers, chitosan, and silk fibroin nanoparticles, during the recycling process for improving the properties of the recycled plastic. The degradation level of the aged polymer and the nature and amount of filler affect the performance of the recycled plastics. The fillers reduce the degradation during the melt reprocessing of PLA previously subjected to severe hydrolysis, thus increasing the intrinsic viscosity of the recycled plastic. A careful selection of the added organic filler lead to recycled plastics with improvements in some key mechanical, thermal, and barrier properties. Thus, the use of organic fillers represents a cost-effective and environmentally sound way for improving the mechanical recycling of bioplastics.

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, silk fibroin-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 vitro for the seven days of the experiment. SIGNIFICANCE: 3D biohybrid retina can be built using silk silkworm fibroin films and hydrogels to be used in cell replacement therapy for AMD and similar retinal neurodegenerative diseases.

Electrospun silk fibroin/TiO2 mats. Preparation, characterization and efficiency for the photocatalytic solar treatment of pesticide polluted water.

The photocatalytic properties of silk fibroin (SF) incorporating TiO2 nanoparticles using an electrospinning technique were examined. Electrospun SF/TiO2 mats were successfully prepared and characterized by different techniques (XRD, FE-SEM, XPS, XDS, FTIR and BET). The photocatalytic efficiency of these materials were assessed by their ability to degrade four pesticides (boscalid, hexythiazox, pyraclostrobin and trifloxystrobin) in water exposed to solar irradiation. The effect of catalyst loading on the disappearance kinetics of the different pesticides was studied in order to determine the maximum degradation efficiency. The degradation rate significantly increases upon adding the TiO2. However, no significant differences (p < 0.05) were observed when the TiO2 loading was increased from 25 to 50 mg for most compounds. Thus, SF mats with 25 mg of TiO2 were selected. Therefore, a new and simple approach to produce materials with photocatalytic activity, safety and potential application in the purification of water contaminated by pesticides has been developed.

Effect of different cocoon stifling methods on the properties of silk fibroin biomaterials.

Stifling treatments are applied to silk cocoons in order to kill the pupae, preventing the emergence of moths and allowing to preserve the silk during long periods of time. All of them involve the application of aggressive steps, such as sun exposure, hot steam from boiling water or hot air, during hours or even days. None of the scientific articles related to silk fibroin biomaterials has previously taken into account this fact in its section of materials and methods. In this work, the consequences of the stifling treatments most commonly used by the silk producing countries and companies are explored in depth, using fibroin films as biomaterial model. The protein degradation (visualised by SDS-PAGE) was dramatically increased in all the fibroin dissolutions produced from stifled cocoons; heavy and light chains of fibroin were specially degraded, reducing their presence along the lanes of the gel compared to the negative control (untreated fresh cocoons). Structural changes are also described for annealed silk fibroin films. The ß-sheet content, analysed by means of infrared spectroscopy, was significantly higher when stifling was performed at higher temperature (70 °C and 85 °C). It is also exposed the impact of the stifling on the mechanical properties of the materials. Tensile strength and strain at break values were detected as significantly lower when this procedure was carried out by means of dry heat (85 °C) and sun exposure. On the other hand, and contrary to expectations, the proliferation of fibroblasts growing on the materials was improved by all the different stifling methods, compared to negative control, being this improvement, especially accentuated, on the films produced with fibroin purified from cocoons treated with dry heat.

Silk fibroin scaffolds seeded with Wharton's jelly mesenchymal stem cells enhance re-epithelialization and reduce formation of scar tissue after cutaneous wound healing.

BACKGROUND: The treatment of extensive and/or chronic skin wounds is a widespread and costly public health problem. Mesenchymal stem cells (MSCs) have been proposed as a potential cell therapy for inducing wound healing in different clinical settings, alone or in combination with biosynthetic scaffolds. Among them, silk fibroin (SF) seeded with MSCs has been shown to have increased efficacy in skin wound healing experimental models. METHODS: In this report, we investigated the wound healing effects of electrospun SF scaffolds cellularized with human Wharton's jelly MSCs (Wj-MSCs-SF) using a murine excisional wound splinting model. RESULTS: Immunohistopathological examination after transplant confirmed the presence of infiltrated human fibroblast-like CD90-positive cells in the dermis of the Wj-MSCs-SF-treated group, yielding neoangiogenesis, decreased inflammatory infiltrate and myofibroblast proliferation, less collagen matrix production, and complete epidermal regeneration. CONCLUSIONS: These findings indicate that Wj-MSCs transplanted in the wound bed on a silk fibroin scaffold contribute to the generation of a well-organized and vascularized granulation tissue, enhance reepithelization of the wound, and reduce the formation of fibrotic scar tissue, highlighting the potential therapeutic effects of Wj-MSC-based tissue engineering approaches to non-healing wound treatment.

Potential use of silkworm gut fiber braids as scaffolds for tendon and ligament tissue engineering.

Tendon and ligament tissue engineering require scaffolds for the treatment of various conditions in the medical field. These must meet requirements such as high tensile strength, biocompatibility, fast and stable repair and a rate of degradation that allows the repair of the damaged tissue. In this work, we propose the use of silkworm gut fiber braids as materials to temporarily replace and repair this type of tissues. The mechanical characterization of the braids made with different number of silk gut fibers is provided, as well as a descriptive analysis of the proliferation and adhesion of cultures of adult human mesenchymal stem cells from bone marrow and fibroblasts (L929) on the braids. As expected, the breaking force increases linearly in the scaffold with the number of fibers, thus being a parameter adaptable to the specific requirements of the tissue to repair and the animal model of study. On the other hand, in all of the cases studied, the values obtained for the elastic modulus of the hydrated fibers were in the range of the ones reported for various human tendons and ligaments. Moreover, the scaffold demonstrated excellent biocompatibility in vitro, allowing the adhesion and proliferation, in the same culture conditions, of the two cell types studied, therefore posing as an ideal candidate to be employed in future in vivo studies that allow elucidating its behavior in the articular environment or extra-articular tendinous areas. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res B Part B: 2019. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2209-2215, 2019.

Silk fibroin nanoparticles as biocompatible nanocarriers of a novel light-responsive CO-prodrug.

[Mn(CO)3(2,2'-bipyridine)(PPh3)](ClO4) (1), a novel photoactive CO-releasing molecule, has been prepared and fully characterized. Besides, silk fibroin nanoparticles (SFNs) have been used, for the first time, as vehicles of 1 leading to the hybrid material 1@SFNs that shows an enhanced CO-delivery.

Production of Curcumin-Loaded Silk Fibroin Nanoparticles for Cancer Therapy.

Curcumin, extracted from the rhizome of Curcuma longa, has been widely used in medicine for centuries due to its anti-inflammatory, anti-cancer, anti-oxidant and anti-microbial effects. However, its bioavailability during treatments is poor because of its low solubility in water, slow dissolution rate and rapid intestinal metabolism. For these reasons, improving the therapeutic efficiency of curcumin using nanocarriers (e.g., biopolymer nanoparticles) has been a research focus, to foster delivery of the curcumin inside cells due to their small size and large surface area. Silk fibroin from the Bombyx mori silkworm is a biopolymer characterized by its biocompatibility, biodegradability, amphiphilic chemistry, and excellent mechanical properties in various material formats. These features make silk fibroin nanoparticles useful vehicles for delivering therapeutic drugs, such as curcumin. Curcumin-loaded silk fibroin nanoparticles were synthesized using two procedures (physical adsorption and coprecipitation) more scalable than methods previously described using ionic liquids. The results showed that nanoparticle formulations were 155 to 170 nm in diameter with a zeta potential of approximately -45 mV. The curcumin-loaded silk fibroin nanoparticles obtained by both processing methods were cytotoxic to carcinogenic cells, while not decreasing viability of healthy cells. In the case of tumor cells, curcumin-loaded silk fibroin nanoparticles presented higher efficacy in cytotoxicity against neuroblastoma cells than hepatocarcinoma cells. In conclusion, curcumin-loaded silk fibroin nanoparticles constitute a biodegradable and biocompatible delivery system with the potential to treat tumors by local, long-term sustained drug delivery.

Silk Fibroin Films for Corneal Endothelial Regeneration: Transplant in a Rabbit Descemet Membrane Endothelial Keratoplasty.

Purpose: Develop a silk fibroin (SF)-based artificial endothelial graft for its use in a rabbit Descemet membrane endothelial keratoplasty (DMEK). Methods: Human and rabbit artificial corneal endothelial grafts were developed through the culture of human and rabbit corneal endothelial cells (CECs) on SF films. Rabbit artificial SF endothelial grafts were transplanted in a DMEK surgery into a rabbit in vivo model. Results: SF artificial endothelial grafts showed the characteristic endothelial markers: zonula occludens (ZO-1) and Na+/K+ ATPase. In a rabbit model of DMEK surgery, SF artificial endothelial graft restored the corneal transparency and thickness at 6 week of follow-up. Anterior segment optical coherence tomography revealed the SF graft as a fully integrated component in the corneal tissue, displaying a similar corneal thickness and endothelial cell count when compared with its healthy contralateral cornea. Histologic analysis showed that the SF artificial endothelial graft was attached and integrated on the surface of the corneal stroma without a significant inflammatory reaction, and rabbit CECs consisted in a monolayer that showed their characteristic markers ZO-1 and Na+/K+ ATPase, suggesting proper intercellular junctions and cellular pump function. Conclusions: We have developed SF films with biological properties that supported the growth of rabbit and human CECs, which showed normal morphology and characteristic markers; and with mechanical properties that allowed its use in a DMEK surgery, proving its in vivo functionality in a rabbit model of endothelial dysfunction.

Electrospun silk fibroin scaffolds coated with reduced graphene promote neurite outgrowth of PC-12 cells under electrical stimulation.

Novel approaches to neural research require biocompatible materials capable to act as electrode structures or scaffolds for tissue engineering in order to stimulate or restore the functionality of damaged tissues. This work offers promising results that indicate the potential use of electrospun silk fibroin (SF) scaffolds coated with reduced graphene oxide (rGO) in this sense. The coated material becomes conductor and electroactive. A complete characterisation of SF/rGO scaffolds is provided in terms of electrochemistry, mechanical behaviour and chemical conformation of fibroin. The excellent biocompatibility of this novel material is proved with cultures of PC-12 cells. The coating with rGO improved the adhesion of cells in comparison with cells growing onto the surface of pure SF scaffolds. Also, the use of SF/rGO scaffolds combined with electrical stimulation promoted the differentiation into neural phenotypes reaching comparable or even superior levels to those obtained by means of the traditional treatment with neural growth factor (NGF).

Impact of Covalent Functionalization on the Aqueous Processability, Catalytic Activity, and Biocompatibility of Chemically Exfoliated MoS2 Nanosheets.

Chemically exfoliated MoS2 (ce-MoS2) has emerged in recent years as an attractive two-dimensional material for use in relevant technological applications, but fully exploiting its potential and versatility will most probably require the deployment of appropriate chemical modification strategies. Here, we demonstrate that extensive covalent functionalization of ce-MoS2 nanosheets with acetic acid groups (∼0.4 groups grafted per MoS2 unit) based on the organoiodide chemistry brings a number of benefits in terms of their processability and functionality. Specifically, the acetic acid-functionalized nanosheets were furnished with long-term (>6 months) colloidal stability in aqueous medium at relatively high concentrations, exhibited a markedly improved temporal retention of catalytic activity toward the reduction of nitroarenes, and could be more effectively coupled with silver nanoparticles to form hybrid nanostructures. Furthermore, in vitro cell proliferation tests carried out with murine fibroblasts suggested that the chemical derivatization had a positive effect on the biocompatibility of ce-MoS2. A hydrothermal annealing procedure was also implemented to promote the structural conversion of the functionalized nanosheets from the 1T phase that was induced during the chemical exfoliation step to the original 2H phase of the starting bulk material, while retaining at the same time the aqueous colloidal stability afforded by the presence of the acetic acid groups. Overall, by highlighting the benefits of this type of chemical derivatization, the present work should contribute to strengthen the position of ce-MoS2 as a two-dimensional material of significant practical utility.

Fabrication of electrospun silk fibroin scaffolds coated with graphene oxide and reduced graphene for applications in biomedicine.

Silk fibroin and graphene are both promising biomaterials described in the bibliography. Hybrid scaffolds combining their properties could be attractive for tissue engineering applications. In this work, a new methodology to produce electrospun fibroin scaffolds coated with graphene materials is provided. The mechanical, electrical and electrochemical properties of the materials attained were characterised. The fibre diameters were measured (from 3.9 to 5.2 µm). The samples coated with reduced grapheme were electronic conductors and electroactive in liquid electrolytes, showing maximum oxidation and reduction (around−0.4 V peak). The chronoamperometric responses showed a reduction shoulder, pointing to the entrance of balancing cations from the solution by nucleation­relaxation: the reaction induced structural changes in the graphene. In order to check the biocompatibility of the materials, they were seeded with L929 fibroblasts. The excellent biocompatibility of silk fibroin meshes was maintained after coating with graphene, being the proliferation results equal in all the treatments 7 days after the seeding (Tukey, p N 0.05).The conductive and electroactive properties of meshes coated with reduced graphene allow the potential application of local electric fields or local ionic currents to cell cultures, biological interfaces or animal models without host response.

Mechanical behaviour and formation process of silkworm silk gut.

High performance silk fibers were produced directly from the silk glands of silkworms (Bombyx mori) following an alternative route to natural spinning. This route is based on a traditional procedure that consists of soaking the silk glands in a vinegar solution and stretching them by hand leading to the so called silkworm guts. Here we present, to the authors' best knowledge, the first comprehensive study on the formation, properties and microstructure of silkworm gut fibers. Comparison of the tensile properties and microstructural organization of the silkworm guts with those of naturally spun fibers allows gain of a deeper insight into the mechanisms that lead to the formation of the fiber, as well as the relationship between the microstructure and properties of these materials. In this regard, it is proved that an acidic environment and subsequent application of tensile stress in the range of 1000 kPa are sufficient conditions for the formation of a silk fiber.

Influence of the protocol used for fibroin extraction on the mechanical properties and fiber sizes of electrospun silk mats.

Silk fibroin (SF) was regenerated using three of the most common protocols described in the bibliography for the dissolution of raw SF (LiBr 9.3M, CaCl2 50 wt.% or CaCl2:EtOH:H2O 1:2:8 in molar ratio). The integrity of regenerated SF in aqueous solution was analyzed by SDS-PAGE and different profiles of degradation were observed depending on the protocol used. This fact was found to affect also the aqueous solubility of the freeze dried protein. These different SFs were used to produce electrospun mats using SF solutions of SF 17 wt.% in 1,1,1,1',1',1'-hexafluoro-2-propanol (HFIP) and significant differences in fiber sizes, elongation and ultimate strength values were found. This work provides a global overview of the manner that different methods of SF extraction can affect the properties of electrospun SF-mats and consequently it should be considered depending on the use they are going to be made for.

A photoactivated nanofiber graft material for augmented Achilles tendon repair.

BACKGROUND AND OBJECTIVE: Suture repair of Achilles tendon rupture can cause infection, inflammation and scarring, while prolonged immobilization promotes adhesions to surrounding tissues and joint stiffness. Early mobilization can reduce complications provided the repair is strong enough to resist re-rupture. We have developed a biocompatible, photoactivated tendon wrap from electrospun silk (ES) to provide additional strength to the repair that could permit early mobilization, and act as a barrier to adhesion formation. STUDY DESIGN/MATERIAL AND METHODS: ES nanofiber mats were prepared by electrospinning. New Zealand white rabbits underwent surgical transection of the Achilles tendon and repair by: (a) SR: standard Kessler suture + epitendinous suture (5-0 vicryl). (b) ES/PTB: a single stay suture and a section of ES mat, stained with 0.1% Rose Bengal (RB), wrapped around the tendon and bonded with 532 nm light (0.3 W/cm(2) , 125 J/cm(2) ). (c) SR + ES/PTB: a combination of (a) and (b). Gross appearance, extent of adhesion formation and biomechanical properties of the repaired tendon were evaluated at Days 7, 14, or 28 post-operatively (n = 8 per group at each time point). RESULTS: Ultimate stress (US) and Young's modulus (E) in the SR group were not significantly different from the ES/PTB group at Days 7 (US, P = 0.85; E, P = 1), 14 (US, P = 0.054; E, P = 1), and 28 (US, P = 0.198; E, P = 0.12) post-operatively. Adhesions were considerably greater in the SR group compared to the ES/PTB group at Days 7 (P = 0.002), 14 (P < 0.0001), and 28 (P < 0.0001). The combination approach of SR + ES/PTB gave the best outcomes in terms of E at 7 (P < 0.016) and 14 days (P < 0.016) and reduced adhesions compared to SR at 7 (P < 0.0001) and 14 days (P < 0.0001), the latter suggesting a barrier function for the photobonded ES wrap. CONCLUSION: Photochemical sealing of a ES mat around the tendon repair site provides considerable benefit in Achilles tendon repair. Lasers Surg. Med. 44: 645-652, 2012. © 2012 Wiley Periodicals, Inc.

Fabrication of conductive electrospun silk fibroin scaffolds by coating with polypyrrole for biomedical applications.

Scaffolds constituted by micro and nanofibers of silk fibroin were obtained by electrospinning. Fibers of fibroin meshes were coated with polypyrrole (pPy) by chemical polymerization; chemical linkages between polymers were observed by SEM and IR spectroscopy. Mechanical resistance of the meshes was improved by polypyrrole coating. Furthermore, coated meshes present a high electroactivity allowing anion storage and delivery during oxidation/reduction reactions in aqueous solutions. Uncoated and pPy coated materials support the adherence and proliferation of adult human mesenchymal stem cells (ahMSCs) or human fibroblasts (hFb). The bioactivity of fibroin mesh overcomes that of the polypyrrole coated meshes.