Preparation and mechanical optimization of a two-layer silk/magnesium wires braided porous artificial nerve guidance conduit.

Peripheral nerve injures have long been a tricky problem in surgery and a feasible treatment is the transplantation of nerve guidance conduits (NGCs). This study presents a two-layer composite NGC with fair mechanical properties and good biocompatibility. The inner layer was made of degummed silk yarns/magnesium wires using braiding technology, and the outer layer was made from mixed solution of silk fibroin/chitosan (SF/CS) using freeze-drying treatment. Orthogonal experimental design was applied to rationally design the braided structural layer and obtain the optimal combination of technical process parameters. Meanwhile, the SF/CS porous outer layer was optimized from three concentrations of SF/CS solution. In vitro and in vivo study suggested that the textile-forming scaffold exhibited good biocompatibility and no toxicity. During 4 weeks' degradation, the skeleton of conduits retained its shape, and magnesium ions released from degraded magnesium wires contributed to sustainable release and uniform dispersion, proliferation and adhesion of Schwann cells, indicating potential approach in the development of NGCs.

Sustainable Antibacterial and Anti-Inflammatory Silk Suture with Surface Modification of Combined-Therapy Drugs for Surgical Site Infection.

Silk sutures with antibacterial and anti-inflammatory functions were developed for sustained dual-drug delivery to prevent surgical site infections (SSIs). The silk sutures were prepared with core-shell structures braided from degummed silk filaments and then coated with a silk fibroin (SF) layer loaded with berberine (BB) and artemisinin (ART). Both the rapid release of drugs to prevent initial biofilm formation and the following sustained release to maintain effective concentrations for more than 42 days were demonstrated. In vitro assays using human fibroblasts (Hs 865.Sk) demonstrated cell proliferation on the materials, and hemolysis was 2.4 ± 0.8%, lower than that required by ISO 10993-4 standard. The sutures inhibited platelet adhesion and promoted collagen deposition and blood vessel formation. In vivo assessments using Sprague-Dawley (SD) rats indicated that the coating reduced the expression of pro-inflammatory cytokines interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α), shortening the inflammatory period and promoting angiogenesis. The results demonstrated that these new sutures exhibited stable structures, favorable biocompatibility, and sustainable antibacterial and anti-inflammatory functions with potential for surgical applications.

Low-Density Silk Nanofibrous Aerogels: Fabrication and Applications in Air Filtration and Oil/Water Purification.

A method was developed to fabricate light, water-insoluble silk fibroin nanofibrous aerogels (SNFAs) through solvent welding of lyophilized silk nanofibrous 3D networks at the junction points while converting silk structures from random-coils to ß-sheets (water insoluble). Aromatic alcohols, especially phenethyl alcohol (PEA), supported robust solvent welding and the structural conversion of silk. PEA vapor treatment was a better approach than solvent infusion to retain volume, density, and mechanical strength of the SNFAs. The mechanical properties of highly orientated SNFAs were superior to randomly distributed fibers. The SNFAs had a low density (3.5 mg/cm3), high hydrophobicity (140.9°), and a porous surface morphology on the individual nanofibers, resulting in high efficiency and selectivity for absorbing particulate matter and oils. Compared with commonly used inorganic aerogels, the SNFAs developed in this study are biocompatible, easily functionalized, environmentally friendly, and low-cost and therefore have potential for air and water purification, biosensors, drug delivery, and tissue engineering.

Control of octreotide release from silk fibroin microspheres.

Poly(d,l-lactide-co-glycolide) (PLGA) microspheres have been used as an injectable depot for prolonged release of octreotide (Sandostatin LAR®), a peptide drug for the treatment of acromegaly and gastrointestinal tumors. However, acylation and incomplete release of the encapsulated octreotide, as well as acidic degradation product-induced inflammation are the major challenges hampering widespread clinical applications of this delivery system. The purpose of this study was to develop a novel octreotide-delivering system utilizing naturally derived biodegradable material, silk fibroin (SF). Octreotide acetate was encapsulated in the SF microspheres with a high loading (8-10 wt%) using polyethylene glycol (PEG)-assisted emulsification method. The octreotide-SF microspheres exhibited a silk I structure (low crystallinity) and burst release in in vitro release studies. Ethanol treatment after microsphere formation significantly increased ß-sheet and silk II structure (high crystallinity) of the microspheres, significantly reducing the burst release and resulting in zero-order sustained release of octreotide over 102 days, and the data could be fit to the diffusion-driven release model. After the ethanol-treated microspheres were intramuscularly injected into rats at low (2 mg/kg) and high (8 mg/kg) octreotide doses, the plasma concentration of octreotide in the high dose group remained high (>50 pg/mL) at day 28 when compared to that of the control (pure drug at low dose) and low dose microsphere group. Interestingly, the plasma concentration for the high dose group at day 56 dramatically increased to >280 pg/mL observed at day 28. The low dose microsphere group showed a similar increase, but at a much lower level. The rebound octreotide level likely reflected degradation of the SF matrix which released tightly bound/trapped octreotide. Therefore, SF microspheres can deliver octreotide over a long period of time with release kinetics and the mechanism different from PLGA microsphere system.

A Biodegradable Stent with Surface Functionalization of Combined-Therapy Drugs for Colorectal Cancer.

In-stent restenosis caused by tumor ingrowth is a major problem for patients undergoing stent placement because conventional stents often lack sustainable antitumor capabilities. The aim of this work is to develop a silk fibroin (SF)-based nanofibrous membrane that is loaded with combined-therapy drugs by using electrospinning technologies, which is further coated on a polydioxanone (PDO) stent and used for the treatment of colorectal cancer (CRC). In order to improve treatment effectiveness, a combination of therapeutic drugs, i.e., curcumin (CUR) and 5-fluorouracil (5-FU), is dissolved into SF solution and then eletrospun onto the surface of the PDO stent. The morphology, secondary structure, and in vitro drug release profiles of the membranes are characterized. The antitumor efficacy is assessed in vitro and in vivo using a human CRC cell line and normal cells, and tumor-bearing nude mice. In vitro and in vivo studies on the nanofibrous memembrane-coating demonstrate improved antitumor effects for the CUR/5-FU dual drug system which can be attributed to cell cycle arrest in the S phase in association with induced apoptosis in tumor cells by blocking signal transducer and activator of transcription3 (Stat3) and nuclear factor kappa beta (NF-kB) signaling pathways, suggesting potential in the treatment of CRC in the future.

Silk Fibroin-Based Fibrous Anal Fistula Plug with Drug Delivery Function.

The aim of this work is to develop a drug-loaded silk fibroin fibrous membrane (DSFM) that can be attached to the surface of an anal fistula plug to improve the treatment of Crohn's disease (CD). Curcumin (CUR) and 5-aminosalicylic acid (5-ASA)-loaded silk fibroin (SF) membranes are coaxially electrospun onto the surface of a braided silk filament plug. The membranes show a predominant structure of random coil and silk I conformation. The concentration of CUR/5-ASA (weight ratio of 1/1) in the SF solution is optimized to 0.4, 0.9, and 1.9 wt%. The morphologies, secondary structures, and in vitro drug release properties of the membranes are examined. Sectional images of fibers in the membranes show core-shell structures. The coaxial electrospinning process does not alter the chemical characteristics of the drugs. The dual-drugs encapsulated in the membranes are released in a steady and sustainable manner, and the cumulative release rate is improved by the increased drug loading. The membranes exhibit no cytotoxicity, thereafter increase the viability of human fibroblasts on the DSFMs. These SF membranes with core-shell structure and functional encapsulation of CUR and 5-ASA should be useful for further studies toward the treatment of CD.

3D Bioprinting of Self-Standing Silk-Based Bioink.

Silk/polyethylene glycol (PEG) hydrogels are studied as self-standing bioinks for 3D printing for tissue engineering. The two components of the bioink, silk fibroin protein (silk) and PEG, are both Food and Drug Administration approved materials in drug and medical device products. Mixing PEG with silk induces silk ß-sheet structure formation and thus gelation and water insolubility due to physical crosslinking. A variety of constructs with high resolution, high shape fidelity, and homogeneous gel matrices are printed. When human bone marrow mesenchymal stem cells are premixed with the silk solution prior to printing and the constructs are cultured in this medium, the cell-loaded constructs maintain their shape over at least 12 weeks. Interestingly, the cells grow faster in the higher silk concentration (10%, w/v) gel than in lower ones (7.5 and 5%, w/v), likely due to the difference in material stiffness and the amount of residual PEG remaining in the gel related to material hydrophobicity. Subcutaneous implantation of 7.5% (w/v) bioink gels with and without printed fibroblast cells in mice reveals that the cells survive and proliferate in the gel matrix for at least 6 week postimplantation. The results suggest that these silk/PEG bioink gels may provide suitable scaffold environments for cell printing and function.

Curcumin-functionalized silk biomaterials for anti-aging utility.

Curcumin is a natural antioxidant that is isolated from turmeric (Curcuma longa) and exhibits strong free radical scavenging activity, thus functional for anti-aging. However, poor stability and low solubility of curcumin in aqueous conditions limit its biomedical applications. Previous studies have shown that the anti-oxidation activity of curcumin embedded in silk fibroin films could be well preserved, resulting in the promoted adipogenesis from human mesenchymal stem cells (hMSCs) cultured on the surface of the films. In the present study, curcumin was encapsulated in both silk fibroin films (silk/cur films) and nanoparticles (silk/cur NPs), and their anti-aging effects were compared with free curcumin in solution, with an aim to elucidate the mechanism of anti-aging of silk-associated curcumin and to better serve biomedical applications in the future. The morphology and structure of silk/cur film and silk/cur NP were characterized using SEM, FTIR and DSC, indicating characteristic stable beta-sheet structure formation in the materials. Strong binding of curcumin molecules to the beta-sheet domains of silk fibroin resulted in the slow release of curcumin with well-preserved activity from the materials. For cell aging studies, rat bone marrow mesenchymal stem cells (rBMSCs) were cultured in the presence of free curcumin (FC), silk/cur film and silk/cur NP, and cell proliferation and markers of aging (P53, P16, HSP70 gene expression and ß-Galactosidase activity) were examined. The results indicated that cell aging was retarded in all FC, silk/cur NP and silk/cur film samples, with the silk-associated curcumin superior to the FC.

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.

Inkjet-assisted layer-by-layer printing of quantum dot/enzyme microarrays for highly sensitive detection of organophosphorous pesticides.

We present a facile fabrication of layer-by-layer (LbL) microarrays of quantum dots (QDs) and acetylcholinesterase enzyme (AChE). The resulting arrays had several unique properties, such as low cost, high integration and excellent flexibility and time-saving. The presence of organophosphorous pesticides (OPs) can inhibit the AChE activity and thus changes the fluorescent intensity of QDs/AChE microscopic dot arrays. Therefore, the QDs/AChE microscopic dot arrays were used for the sensitive visual detection of OPs. Linear calibration for parathion and paraoxon was obtained in the range of 5-100 µg L(-1) under the optimized conditions with the limit of detection (LOD) of 10 µg L(-1). The arrays have been successfully used for detection of OPs in fruits and water real samples. The new array was validated by comparison with conventional high performance liquid chromatography-mass spectrometry (HPLC-MS).

Adhesion Prevention after Laminectomy Using Silk-Polyethylene Glycol Hydrogels.

Laminectomy is a common operation in spine surgery to reduce spinal cord and nerve pressure. However, scar tissues often form in the spinal canal and adhere to the dura surface, resulting in low back pain postsurgery. In the present study, biodegradable silk-polyethylene glycol (PEG) hydrogels are evaluated for adhesion prevention after laminectomies in New Zealand rabbits, with nondegradable expanded polytetrafluoroethylene (ePTFE) membranes and saline as controls. No significant difference among the three groups is observed within 2 weeks. Silk is fully degraded within 6 weeks, leaving a gap separating the scar tissue and the dura mater. Severe dural scar adhesion form in the saline control group after 8 weeks, while no or mild adhesion is observed in the ePTFE membrane and silk-PEG hydrogel samples. Human dermal fibroblasts (HS-865-SK cells) are cultured in the silk-PEG hydrogel extracts and on top of gel surfaces. Compared to the controls of tissue culture plate (no silk) and sonicated silk hydrogels (no PEG), the proliferation of fibroblasts in both conditions is significantly reduced initially but resumes after 120 h, suggesting the surface properties of the hydrogels and local, temporal release of PEG accounts for the adhesion prevention observed in vivo in this study.

Curcumin-functionalized silk materials for enhancing adipogenic differentiation of bone marrow-derived human mesenchymal stem cells.

Curcumin, a natural phenolic compound derived from the plant Curcuma longa, was physically entrapped and stabilized in silk hydrogel films, and its influence on human bone marrow-derived mesenchymal stem cells (hBMSC) was assessed related to adipogenic differentiation. The presence of curcumin significantly reduced the silk gelation time and changed the porous morphology of gel matrix, but did not change the formation of the silk beta-sheet structure. Based on spectrofluorimetric analysis, curcumin most likely interacted with hydrophobic residues in silk, interacting with the beta-sheet domains formed in the hydrogels. The antioxidant activity of silk film-associated curcumin remained functional over at least one month in both the dry and hydrated state. Negligible curcumin was released from silk hydrogel films over 48 h incubation in aqueous solution. For hBMSC cultured on silk films containing more than 0.25 mg ml(-1) curcumin, cell proliferation was inhibited, while adipogenesis was significantly promoted based on transcripts as well as Oil Red O staining. When hBMSC were cultured in media containing free curcumin, both proliferation and adipogenesis of hBMSC were inhibited when curcumin concentrations exceeded 5 µM, which is more than 1000 times higher than the level of curcumin released from the films in aqueous solution. Thus, silk film-associated curcumin exhibited different effects on hBMSC proliferation and differentiation compared with curcumin in solution.

Nanostructured photoelectrochemical biosensor for highly sensitive detection of organophosphorous pesticides.

A sensitive photoelectrochemical (PEC) biosensor for detection of organophosphorus pesticides (OPs) using the nanocomposite of CdSe@ZnS quantum dots (QDs) and graphene deposited on the ITO coated glass electrode as a photoactive electrode is presented. The integration of CdSe@ZnS/graphene nanocomposite with biomolecules acetylcholinesterase (AChE) as a biorecognition element yields a novel biosensing platform. Under visible light irradiation, the AChE-CdSe@ZnS/graphene nanocomposite can generate a stable photocurrent and the photocurrent is found to be inversely dependent on the concentration of OPs. Under the optimal experimental conditions, the photocurrents were proportional to the logarithm of paraoxon and dichlorvos within the concentration range of 10(-12)-10(-6) M. The detection limits (LOD) of the proposed biosensor for paraoxon and dichlorvos are as low as 10(-14) M and 10(-12) M. The photoelectrochemical biosensor shows good sensitivity, reproducibility, stability, and could be successfully applied to detection of OPs in real fruit samples.