Hybrid Bionanocomposite Containing Magnesium Hydroxide Nanoparticles Embedded in a Carboxymethyl Cellulose Hydrogel Plus Silk Fibroin as a Scaffold for Wound Dressing Applications.

Based on the promising biomedical developments in wound healing strategies, herein, a new nanobiocomposite scaffold was designed and presented by incorporation of carboxymethyl cellulose hydrogels prepared using epichlorohydrin as a cross-linking agent (CMC hydrogel), a natural silk fibroin (SF) protein, and magnesium hydroxide nanoparticles (Mg(OH)2 NPs). Biological evaluation of the CMC hydrogel/SF/Mg(OH)2 nanobiocomposite scaffold was conducted via in vitro cell viability assays and in vivo assays, red blood cell hemolysis, and antibiofilm assays. Considering the cell viability percentage of Hu02 cells (84.5%) in the presence of the prepared nanobiocomposite after 7 days, it was indicated that this new nanoscaffold was biocompatible. The signs of excellent hemocompatibility and the high antibacterial activity were observed due to the low-point hemolytic effect (8.3%) and high-level potential in constraining the P. aeruginosa biofilm formation with a low OD value (0.13). Moreover, in vivo wound healing assay results indicated that the wound healing method was faster in mice treated with the prepared nanobiocomposite scaffold (82.29%) than the control group (75.63%) in 12 days. Apart from the structural characterization of the CMC hydrogel/SF/Mg(OH)2 nanobiocomposite through FTIR, EDX, FESEM, and TG analyses, compressive mechanical tests, contact angle, porosity, and swelling ratio studies indicated that the combination of the CMC hydrogel structure with SF protein and Mg(OH)2 NPs could significantly impact Young's modulus (from 11.34 to 10.14 MPa), tensile strength (from 299.35 to 250.78 MPa), elongation at break (12.52 to 12.84%), hydrophilicity, and water uptake capacity (92.5%).

The relationship between crosslinking structure and silk fibroin scaffold performance for soft tissue engineering.

Biologically active scaffolds with tunable mechano- and bio-performance remain desirable for soft tissue engineering. Previously, highly elastic and robust silk fibroin (SF) scaffolds were prepared via cryogelation. In order to get more insight into the role of ethylene glycol diglycidyl ether (EGDE) on the structure and properties of SF scaffolds, we investigated the fate of SF scaffolds with different usages of the crosslinking agent in vitro and in vivo. Although SF scaffolds with varied EGDE contents showed similar micro-morphology, increasing EGDE from 1 mmol/g to 5 mmol/g resulted in firstly increased and later decreased content of ß-sheet conformation, and linearly increased tensile modulus and decreased elasticity. The dual-crosslinked SF scaffolds with EGDE up to 5 mmol/g did not show in vitro cytotoxicity for NIH3T3 fibroblasts. In vivo subcutaneous implantation of SF scaffolds with <3 mmol/g EGDE displayed excellent degradation behavior and tissue ingrowth after 28 days of implantation. However, with ≥3 mmol/g EGDE, SF scaffolds exhibited obvious post-implantation foreign body reactions, probably associated with slow degradation due to excess chemical crosslinks and less mechanical compatibility. These results suggest that an appropriate dosage of crosslinking agent was critical to achieve balanced mechanical properties, degradability in vivo and immuno-properties of the SF scaffold platform for soft tissue engineering.

Toxicological assessment and food allergy of silk fibroin derived from Bombyx mori cocoons.

Recent studies have demonstrated silk fibroin protein's (SF) ability to extend the shelf life of foods by mitigating the hallmarks of spoilage, namely oxidation and dehydration. Due to the potential for this protein to become more widespread, its safety was evaluated comprehensively. First, a bacterial reverse mutation test (Ames test) was conducted in five bacterial strains. Second, an in vivo erythrocyte test was conducted with Sprague Dawley rats at doses up to 1,000mg/kg-bw/day. Third, a range-finder study was conducted with Sprague Dawley rats at the highest consumption amount given solubility and oral gavage volume constrains (500mg/kg-bw/day). Fourth, a 28-day sub-chronic study in Sprague Dawley rats was conducted with the high dose set at 500mg/kg-bw/day, as limited by solubility of the protein in a single-gavage per-day study. Fifth, an in vitro pepsin digestion assay was performed to assess the potential for protein allergenicity. Sixth, allergenic potential was further assessed using liquid chromatography-mass spectroscopy for detection of allergenic insect proteins. Seventh, the SF protein sequences were subjected to bioinformatic analyses. Together, these studies raise no mutagenic, genotoxic, toxicological, or allergenic concerns with the oral consumption of silk fibroin.

The effect of calcium phosphate and silk fibroin nanofiber tuning on properties of calcium sulfate bone cements.

Calcium sulfate (CS) bone cements have been used as bone substitutes for a long time, but their clinical use is currently limited due to their rapid degradation rate and brittleness. This work aimed to study the effect of α-tricalcium phosphate (α-TCP) and silk fibroin nanofibers (SFF) on CS bone cements. The bone cements were prepared from α-CS hemihydrate (α-CSH), calcium sulfate dihydrate (CSD; as a setting accelerator) and varying α-TCP contents (0%, 5%, 10%, 15%, 20% and 25%), with SFF solution or deionized water as the solidification solution at the same liquid/solid ratio. Scanning electron microscopy, particle size distribution, x-ray diffraction and Fourier transform infrared spectroscopy were used to measure the composition and characterize the properties of the materials. The compressive strength, setting time and weight loss rate of samples were also tested. Cytotoxicity was evaluated by a Cell Counting Kit-8 assay. The results suggest that the tuning of α-TCP and SFF has an important role in determining the compressive strength and degradation rate of CS bone cements, and the properties could be changed by varying the content of α-TCP. Moreover, cell experiments showed no toxicity of the samples towards MC3T3 cells. Thus, the materials prepared from α-CSH, CSD, α-TCP and SFF in this work could provide the basis for research into CS-based bone repair materials.

Tissue-Adhesive Paint of Silk Microparticles for Articular Surface Cartilage Regeneration.

Current biomaterials and tissue engineering techniques have shown a promising efficacy on full-thickness articular cartilage defect repair in clinical practice. However, due to the difficulty of implanting biomaterials or tissue engineering constructs into a partial-thickness cartilage defect, it remains a challenge to provide a satisfactory cure in joint surface regeneration in the early and middle stages of osteoarthritis. In this study, we focused on a ready-to-use tissue-adhesive joint surface paint (JS-Paint) capable of promoting and enhancing articular surface cartilage regeneration. The JS-Paint is mainly composed of N-(2-aminoethyl)-4-(4-(hydroxymethyl)-2-methoxy-5-nitrosophenoxy) butanamide (NB)-coated silk fibroin microparticles and possess optimal cell adhesion, migration, and proliferation properties. NB-modified silk fibroin microparticles can directly adhere to the cartilage and form a smooth layer on the surface via the photogenerated aldehyde group of NB reacting with the -NH2 groups of the cartilage tissue. JS-Paint treatment showed a significant promotion of cartilage regeneration and restored the smooth joint surface at 6 weeks postsurgery in a rabbit model of a partial-thickness cartilage defect. These findings revealed that silk fibroin can be utilized to bring about a tissue-adhesive paint. Thus, the JS-Paint strategy has some great potential to enhance joint surface regeneration and revolutionize future therapeutics of early and middle stages of osteoarthritis joint ailments.

Silk fibroin/hydroxyapatite composite membranes: Production, characterization and toxicity evaluation.

Alloplastic materials based on biopolymers such as silk fibroin (SF) have provided the synthesis of excellent biomaterials for bone repair. The aim of the present study was to produce SF membranes associated to hydroxyapatite (HA) and evaluate their physicochemical characteristics and the toxicity potential. After obtaining the SF, the HPLC was executed to verify the elimitation of serecin, a toxic protein of the silk, and the cytotoxicity assay was assessed in the subtances from the SF processing. SF and SF-HA membranes were evaluated by SEM, EDS, FTIR, mechanical properties and toxicity (cytotoxicity, genotoxicity and mutagenic effects). The serecin was elimined in the SF process, and its cytotoxicity was confirmed. SF and SF-HA membranes presented interesting results based on the physicochemical characterization. SF membrane showed cytotoxic, genotoxic and mutagenic effects. In conclusion, SF and SF-HA membranes presented adequate mechanical resistance to act respectively as wound healing or bone filling materials, and they were hydrophilic. SF-HA membrane did not present any toxic potential and allowed cell adhesion and proliferation. The unexpected cyto/genotoxicity and mutagenic effect of SF evidenced the importance of investigating the toxic potential of biomaterials, mainly those in contact with human body for prolonged time.

Synthesis and assessment of sodium alginate-modified silk fibroin microspheres as potential hepatic arterial embolization agent.

Transcatheter arterial chemoembolization (TACE) is well known as an effective treatment for hepatocellular carcinoma (HCC). In the present study, a novel embolic agent of sodium alginate (SA)-modified silk fibroin (SF) microspheres was successfully prepared by emulsifying cross-linking method. The SA-modified SF microspheres were evaluated for the ability of embolization by investigating the morphology, particle size, swelling ratio, degradation, cytotoxicity, blood compatibility, and in vivo embolization. The results found that SA-modified SF microspheres had smooth surfaces and good sphericity. Swelling ratio of the microspheres can meet the requirements of arterial embolic agent and have pH and temperature sensitivity. Furthermore, hemolytic and anticoagulant studies have proved that the microspheres have good blood compatibility. Cytotoxicity tests indicated that the microspheres could promote the proliferation of fibroblasts and HUVEC. In vivo embolization evaluation of microspheres revealed that the arteries could be embolized by SA-modified SF microspheres in 3 weeks. The ability of drug loading and releasing of microspheres was proved by the controlled release profile of Adriamycin hydrochloride. The findings indicated that the SA-modified SF microspheres can be used as a potentially biodegradable arterial embolic agent for liver cancer therapy.

Smart textiles in wound care: functionalization of cotton/PET blends with antimicrobial nanocapsules.

Management of infected wounds is one of the most costly procedures in the health care sector. Burn wounds are of significant importance due to the high infection risk that can possibly lead to severe consequences such as sepsis. Because antibiotic wound treatments have caused increasing antibiotic resistance in bacteria, there is currently a strong need for alternative strategies. Therefore, we developed new antimicrobial wound dressings consisting of pH-responsive human serum albumin/silk fibroin nanocapsules immobilized onto cotton/polyethylene terephthalate (PET) blends loaded with eugenol, which is an antimicrobial phenylpropanoid. Ultrasound-assisted production of eugenol-loaded nanocapsules resulted in particle sizes (hydrodynamic radii) between 319.73 ± 17.50 and 574.00 ± 92.76 nm and zeta potentials ranging from -10.39 ± 1.99 mV to -12.11 ± 0.59 mV. Because recent discoveries have indicated that the sweat glands contribute to wound reepithelialisation, release studies of eugenol were conducted in different artificial sweat formulas that varied in pH. Formulations containing 10% silk fibroin with lower degradation degree exhibited the highest release of 41% at pH 6.0. After immobilization, the functionalized cotton/PET blends were able to inhibit 81% of Staphylococcus aureus and 33% of Escherichia coli growth. Particle uniformity, silk fibroin concentration, and high surface-area-to-volume ratio of the produced nanocapsules were identified as the contributing factors leading to high antimicrobial activities against both strains. Therefore, the production of antimicrobial textiles using nanocapsules loaded with an active natural compound that will not contribute to antibiotic resistance is seen as a potential future alternative to commercially available antiseptic wound dressings.

Mechanically robust and flexible silk protein/polysaccharide composite sponges for wound dressing.

The cutaneous tissue contains cellular protein and polysaccharide components which together maintain the functionality of the tissue. In this study, silk fibroin (SF) and konjac glucomannan (KGM) were physically crosslinked to form biocompatible protein/polysaccharide sponges with tunable mechanical properties for wound dressing application. The pore structure of sponges can be adjusted by changing blend ratio of SF/KGM, forming homogeneous interconnected pore structure. FTIR and Raman results revealed the intermolecular interaction between SF and KGM, suggesting the formation of interpenetrating polymer network after ethanol/ammonium hydroxide treatment. Raising KGM content significantly enhanced water-absorption, water-retention abilities, and compression strength of porous sponges. Especially, the composite sponges possessed a similar compressive modulus with native skin tissue, showing a matched flexibility for wound treatment. Moreover, the cell viability results based on human dermal fibroblast cells demonstrated that the sponge showed excellent biocompatibility for cell adhesion and proliferation. Therefore, due to the strong water-absorption capacity, moist environment, similar compressive modulus with skin tissue and excellent biocompatibility, the composite sponges have potential application in wound dressings.

Preparation and characterisation of a novel silk fibroin/hyaluronic acid/sodium alginate scaffold for skin repair.

To mimic the natural structure of tissue extracellular matrix, a novel silk fibroin (SF)/hyaluronic acid (HA)/sodium alginate (SA) composite scaffold (92% in porosity) was prepared by freeze-drying. Fourier-transform infrared spectroscopy and Raman spectra indicated interactions among SF, HA, and SA molecules. Scanning electron microscopy showed that the prepared SF/HA/SA scaffold had soft, elastic characteristics, with an average pore diameter of 93 µm. Mechanical property, thermogravimetric analyses and degradation results indicated that the SF/HA/SA scaffold had good physical stability in body fluid and mechanical movement-related environments. Cell proliferation, morphological, and live-dead analyses showed that NIH-3T3 fibroblast cells were better able to attach, grow, and proliferate on the SF/HA/SA scaffold compared with SF, SF/HA, and SF/SA scaffolds. We evaluated the wound healing effects in a rat full-thickness burn model. The hematoxylin-eosin (H&E) and Masson's trichrome staining results from SF/HA/SA scaffold showed that improved re-epithelialization, enhanced extracellular matrix remodeling. Our findings showed that the prepared SF/HA/SA scaffold can provide a potential way as a wound dressing for skin repair.

Evaluation of the toxicity effects of silk fibroin on human lymphocytes and monocytes.

Silk fibroin nanoparticles (SFNPs) as a natural polymer have been utilized in biomedical applications such as suture, tissue engineering-based scaffolds, and drug delivery carriers. Since there is little data regarding the toxicity effects on different cells and tissues, we aimed to determine the toxicity mechanisms of SFNPs on human lymphocytes and monocytes based on reliable methods. Our results showed that SFNPs (0.5, 1, and 2 mg/mL) induced oxidative stress via increasing reactive oxygen species production, mitochondrial membrane potential (∆Ψ) collapse, which was correlated to cytochrome c release and Adenosine diphosphate (ADP)/Adenosine tri phosphate (ATP) ratio increase as well as lysosomal as another toxicity mechanism, which led to cytosolic release of lysosomal digestive proteases, phosphor lipases, and apoptosis signaling. Taken together, these data suggested that SFNPs toxicity was associated with mutual mitochondrial/lysosomal cross-talk and oxidative stress on human lymphocytes and monocytes with activated apoptosis signaling.

Highly water-absorbing silk yarn with interpenetrating network via in situ polymerization.

Silk was modified via in situ polymerization of two monomers acrylamide and sodium acrylate by swelling in an effective LiBr dissolution system. Swelling of natural silks in LiBr solutions of low concentration was clearly observed under optical microscope, and their conformational changes were revealed by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. Dissolution tests and FTIR spectra of these modified silks suggested the presence of interpenetrating network of polyacrylamide and poly(sodium acrylate) in the silk yarns. These modified silks exhibited superior water absorption to that of raw silk and greatly improved mechanical properties in both dry and wet states. These novel modified silks also showed low cytotoxicity towards skin keratinocytes, having potential applications in biomedical textiles. This modification method by in situ polymerization after swelling in LiBr provides a new route to highly enhance the properties and performance of silk for various applications.

Preparation and hemostatic property of low molecular weight silk fibroin.

Effective hemorrhage control becomes increasingly significant in today's military and civilian trauma, while the topical hemostats currently available in market still have various disadvantages. In this study, three low molecular weight silk fibroins (LMSF) were prepared through hydrolysis of silk fibroin in a ternary solvent system of CaCl2/H2O/EtOH solution at different hydrolysis temperatures. Fourier transform infrared spectroscopy analysis showed that the content of ß sheet structure in the LMSF decreased with the increase in hydrolysis temperature. The results of thromboelastographic and activated partial thromboplastin time methods showed that the LMSF hydrolyzed at 50 °C can significantly strengthen the coagulation in blood and activate the intrinsic pathway of coagulation cascade. In the murine hepatic injury model, the LMSF hydrolyzed at 50 °C can promote the blood clotting and decrease the blood loss and bleeding time. Based on these results, it can be suggested that the developed LMSF has the excellent hemostatic effect and may be a promising material in clinical hemostatic application.

Nano/micro hybrid scaffold of PCL or P3HB nanofibers combined with silk fibroin for tendon and ligament tissue engineering.

A novel biodegradable nano/micro hybrid structure was obtained by electrospinning P3HB or PCL nanofibers onto a twisted silk fibroin (SF) structure, with the aim of fabricating a suitable scaffold for tendon and ligament tissue engineering. The electrospinning (ES) processing parameters for P3HB and PCL were optimized on 2D samples, and applied to produce two different nano/micro hybrid constructs (SF/ES-PCL and SF/ES-P3HB).Morphological, chemico-physical and mechanical properties of the novel hybrid scaffolds were evaluated by SEM, ATR FT-IR, DSC, tensile and thermodynamic mechanical tests. The results demonstrated that the nanofibers were tightly wrapped around the silk filaments, and the crystallinity of the SF twisted yarns was not influenced by the presence of the electrospun polymers. The slightly higher mechanical properties of the hybrid constructs confirmed an increase of internal forces due to the interaction between nano and micro components. Cell culture tests with L929 fibroblasts, in the presence of the sample eluates or in direct contact with the hybrid structures, showed no cytotoxic effects and a good level of cytocompatibility of the nano/micro hybrid structures in term of cell viability, particularly at day 1. Cell viability onto the nano/micro hybrid structures decreased from the first to the third day of culture when compared with the control culture plastic, but appeared to be higher when compared with the uncoated SF yarns. Although additional in vitro and in vivo tests are needed, the original fabrication method here described appears promising for scaffolds suitable for tendon and ligament tissue engineering.

Effects of sterilization methods on the physical, chemical, and biological properties of silk fibroin membranes.

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. Sterilization is a fundamental step in biomaterials processing and it must not jeopardize the functionality of medical devices. The aim of this study was to analyze the influence of different sterilization methods in the physical, chemical, and biological characteristics of dense and porous silk fibroin membranes. Silk fibroin membranes were treated by several procedures: immersion in 70% ethanol solution, ultraviolet radiation, autoclave, ethylene oxide, and gamma radiation, and were analyzed by scanning electron microscopy, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, tensile strength and in vitro cytotoxicity to Chinese hamster ovary cells. The results indicated that the sterilization methods did not cause perceivable morphological changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and FTIR results.

Purification and cytotoxicity of tag-free bioengineered spider silk proteins.

Bioengineered spider silk-like proteins can serve as biomaterials for various biomedical applications. These proteins can be assembled in several morphological forms such as films, microcapsules, spheres, fibers, gels, and scaffolds. However, crucial points for recombinant spider silks for human use are toxicity and immunogenicity. To assess this issue, two bioengineered spider silk proteins composed of different numbers of repetitive motifs of the consensus repeats from spidroin-1 from Nephila clavipes (15X and 6X) were cloned and expressed in Escherichia coli. The proteins were free of tag sequence and were purified using two methods based on (1) thermal and (2) organic acid resistance of the spider silks. The soluble spider silk proteins were not cytotoxic and did not activate macrophages over a wide range of concentrations, except when present at the highest concentration. Films made of the different silk variants supported the growth of the cells. Based on these data, and as the biodegradation rate of silk is very slow, the bioengineered spider silks are presumed safe biomaterials for biomedical applications.

Drug loading and release on tumor cells using silk fibroin-albumin nanoparticles as carriers.

Polymeric and biodegradable nanoparticles are frequently used in drug delivery systems. In this study silk fibroin-albumin blended nanoparticles were prepared using the desolvation method without any surfactant. These nanoparticles are easily internalized by the cells, reside within perinuclear spaces and act as carriers for delivery of the model drug methotrexate. Methotrexate loaded nanoparticles have better encapsulation efficiency, drug loading ability and less toxicity. The in vitro release behavior of methotrexate from the nanoparticles suggests that about 85% of the drug gets released after 12 days. The encapsulation and loading of a drug would depend on factors such as size, charge and hydrophobicity, which affect drug release. MTT assay and conjugation of particles with FITC demonstrate that the silk fibroin-albumin nanoparticles do not affect the viability and biocompatibility of cells. This blended nanoparticle, therefore, could be a promising nanocarrier for the delivery of drugs and other bioactive molecules.

Cytotoxicity and endothelial cell adhesion of lyophilized and irradiated bovine pericardium modified with silk fibroin and chitosan.

Grafts of biological tissues have been used since the 1960s as an alternative to the mechanical heart prostheses. Nowadays, the most consolidated treatment to bovine pericardial (BP) bioprostheses is the crosslinking with glutaraldehyde (GA), although GA may induce calcification in vivo. In previous work, our group demonstrated that electron beam irradiation applied to lyophilized BP in the absence of oxygen promoted crosslinks among collagen fibers of BP tissue. In this work, the incorporation of silk fibroin (SF) and chitosan (CHIT) in the BP not treated with GA was studied. The samples were irradiated and then analyzed for their cytotoxicity and the ability of adhesion and growth of endothelial cells. Initially, all samples showed cytotoxicity. However, after a few washing cycles, the cytotoxicity due to acetic acid and ethanol residues was removed from the biomaterial making it suitable for the biofunctional test. The samples modified with SF/CHIT and electron beam irradiated favored the adhesion and growth of endothelial cells throughout the tissue.

Freeze-gelled silk fibroin protein scaffolds for potential applications in soft tissue engineering.

Recently tissue engineering has escalated much interest in biomedical and biotechnological applications. In this regard, exploration of new and suitable biomaterials is needed. Silk fibroin protein is used as one of the most preferable biomaterials for fabrication of scaffolds and several new techniques are being adopted to fabricate silk scaffolds with greater ease, efficiency and perfection. In this study, a freeze gelation technique is used for fabrication of silk fibroin protein 3D scaffolds, which is both time and energy efficient as compared to the conventional freeze drying technique. The fabricated silk fibroin freeze-gelled scaffolds are evaluated micro structurally for morphology with scanning electron microscopy which reveals relatively homogeneous pore structure and good interconnectivity. The pore sizes and porosity of these scaffolds ranges between 60-110µm and 90-95%, respectively. Mechanical test shows that the compressive strength of the scaffolds is in the range of 20-40kPa. The applicability to cell culture of the freeze gelled scaffolds has been examined with human keratinocytes HaCat cells which show the good cell viability and proliferation of cells after 5 days of culture suggesting the cytocompatibility. The freeze-gelled 3D scaffolds show comparable results with the conventionally prepared freeze dried 3D scaffolds. Thus, this technique may be used as an alternative method for 3D scaffolds preparation and may also be utilized for tissue engineering applications.

Differences in cytotoxicity of ß-sheet peptides originated from silk and amyloid ß.

The relationships between amino acid sequence, nano-assemblies, and cytotoxicity to neuron cytotoxicity were investigated using ß-sheet-forming peptides from Araneus ventricosus spider silk, and amyloid forming peptides Aß(12-28) (ß1), Aß(28-42) (ß2), and full-length Aß(1-42). Although silk derived peptides formed nano-assemblies, nanofilaments, and nanofibrils with ß-sheet contents raging from 24 to 40%, they showed no significant cytotoxicity to neurons. In contrast, nano-assemblies and nanofibrils formed from Aß peptides with high ß-sheet content demonstrated cytotoxicity to the neurons. These differences in cell response between the silk ß-sheets and Aß peptides indicate that the general propensity to form beta sheets and form nanostructures is not sufficient to predict cytotoxicity, while surface charges of the assemblies are significant factors that impact cytotoxicity.