Fabricated Silk Fibroin Research Articles

Crystallization of Calcium Deficient Hydroxyapatite Nanocrystals on Woven Silk Fibroin Fabric via Precipitation Process.

A high bioactivity calcium deficient hydroxyapatite (Ca-def HAp) nanocrystal crystallized on a high strength woven silk fibroin (SF) fabric was characterized with the aim to utilize their advantages for the future bone implant applications. Apatite crystal was grown on SF fiber templates by precipitation reaction of Ca(NO₃)₂ prepared from chicken eggshells in (NH₄)₃PO₄ solutions, adjusted pH by NH₄OH solution. In the apatite crystallization process, (NH₄)₃PO₄ solutions prepared at different pH values were poured into the glass dishes containing SF fiber templates/Ca(NO₃)₂ solution, allowed reaction time of only a minute. The results had shown that, the Ca/P ratio of the crystals grown on SF fiber templates were increased through increasing in initial pH value of reaction. Its morphology had been changed from the plate-shaped to particle-shaped crystals. The plainwoven SF fabric was chosen as the suitable template to grow Ca-def HAp crystal for its much higher tensile strength (840 MPa), as compared to 470 MPa for the twill-woven SF fabric. The Cadef HAp nanocrystals on plain-woven SF fabric were confirmed the Ca/P ratio at 1.52 with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The clusters size of smaller than 100 nm Ca-def HAp nanocrystals on plain-woven SF fabric also signified the high surface area of structure which are better for cell attachment, growth and differentiation. Accordingly, the overall result indicated that the composite of combining Ca-def HAp nanocrystals on plain-woven SF fabric had been successfully prepared by the rapid precipitation. The method offers a simpler setup and a shorter processing time and the Ca-def HAp on silk possess a higher tensile strength, suitable for development for future bone implant in the high load bearing regions.

Silk fibroin nanofibrous mats for visible sensing of oxidative stress in cutaneous wounds.

Wound healing is of major clinical concern and is constantly being explored for early restoration and enhanced recovery. While the etiology of the wound healing is multifactorial, high inflammation and increased oxidative stress which results in chronic inflammation, endothelial dysfunction and collagen degradation, delay the overall healing process. Thus, visual sensing of the oxidative stress would be highly informative in the successful implementation of wound healing therapies based on specific requirements. In this study, electrospinning was used to fabricate silk fibroin nanofibrous mats infused with Amplex red capable of detecting hydrogen peroxide, a reactive oxygen molecule. These mats produced a visible change in color with the limit of detection at 1 μM H2O2 concentration. In vivo studies carried out in diabetic mice with impaired wounds also displayed a visible change in color of the mats infused with Amplex red within 24 hours. These electrospun silk fibroin nanofibrous Amplex infused mats has the potential to enable a futuristic platform where decisions can be made for enhanced wound healing therapy.

Fabrication and characterization of conductive silk fibroin–gold nanocomposite films

This research proposes a one-step fabrication of novel conducting silk fibroin (SF) nanocomposites (NCs) in which in situ generation of nanoparticles from their precursor are achieved. Here, the gold salt (HAuIIICl4·H2O) is reduced to gold nanoparticles (AuNPs) using SF, a renewable natural biopolymer, and SF–AuNPs NCs are developed via solution casting method. The optical absorption spectra recorded using the UV–Visible spectroscopy (UV–Vis) witnesses the generation of anisotropic particles by showing two absorption bands. Also, the calculated optical band gap energy (Eg) value decreases from 4.2 to 2.4 eV with the increasing concentration of AuNPs. The X-ray diffraction (XRD) profile of the NCs confirms the presence of AuNPs. The scanning electron microscopy (SEM) micrographs clearly admit the successful formation of AuNPs in the host polymer matrix with homogeneous dispersion. The size and evolution of the different shaped AuNPs are confirmed by the transmission electron microscopy (TEM) study. An increase in DC conductivity from 1.48 × 10−9 to 7.12 × 10−9 S/cm and decrease in frequency-dependent dielectric constant are observed with the increase in AuNPs. The obtained result suggests that the insulating behaviour of the SF can be effectively changed into a conducting nature with the in situ creation of AuNPs in the SF matrix.

Mimicked scaffolds based on coated silk woven fabric with gelatin and chitosan for soft tissue defect in oral maxillofacial area.

Soft tissue defects in the oral maxillofacial area are critical problems for many patients and, in some cases, patients require an operation coupled with a performance scaffold substitution. In this research, mimicked anatomical scaffolds were constructed using gelatin- and chitosan-coated woven silk fibroin fabric. The morphologies, crystals, and structures were observed and then characterized using scanning electron microscopy, X-ray diffraction, and differential scanning calorimetry, respectively. Physical performance was evaluated from the swelling behavior, mechanical properties, and biodegradation, while the biological performance was tested with fibroblasts and keratinocytes, after which cell proliferation, viability, and histology were evaluated. The results revealed that a coated woven silk fibroin fabric displayed a crystal structure of silk fibroin with amorphous gelatin and chitosan layers. Also, the coated fabrics contained residual water within their structure. The physical performance of the coated woven silk fibroin fabric with gelatin showed suitable swelling behavior and mechanical properties along with acceptable biodegradation for insertion at a defect site. The biological performances including cell proliferation, viability, and histology were suitable for soft tissue reconstruction at the defect sites. Finally, the results demonstrated that mimicked anatomical scaffolds based on a gelatin layer on woven silk fibroin fabric had the functionality that was promising for soft tissue construction in oral maxillofacial defect.

Dual-Drug-Loaded Silk Fibroin/PLGA Scaffolds for Potential Bone Regeneration Applications

Developing scaffold materials with excellent biocompatibility, mechanical properties, and controlled drug release properties is vital to tissue engineering. In this study, we fabricated silk fibroin (SF)/poly(lactide-co-glycolide) (PLGA) nanofiber scaffolds containing recombinant human bone morphogenetic protein 2 (rhBMP2) and dexamethasone (DXM) via coaxial electrospinning, which were used in in vitro bone formation with rat bone marrow mesenchymal stem cells (rBMSCs). An in vitro drug release study was adopted to evaluate the sustained release potential of the core-shell structured nanofibers. Furthermore, we detected the potential of the SF/PLGA nanofiber membrane in vitro. In vitro studies showed that rhBMP2 still remained active on the nanofiber membrane. In addition, the dual-drug-loaded nanofiber membrane showed an early burst release of DXM and late sustained release of rhBMP2. rhBMP2 and DXM exhibited strong osteogenic differentiation potential when they acted on rBMSCs. Therefore, the SF/PLGA nanofiber membrane loaded with rhBMP2 and DXM has great potential for the enhancement of bone regeneration.

Femtosecond direct laser writing of silk fibroin optical waveguides

Silk fibroin is an abundant natural polymer, which in the last years has been proposed as an attractive material for high-technological applications, offering also new opportunities for photonics and optoelectronics. In this context, the design of optical waveguides based on silk fibroin is of great interest because of their ability to direct light in a controlled manner. In this work, we demonstrate the use of femtosecond direct laser writing (fs-DLW) to fabricate silk fibroin waveguides. Such waveguides were characterized by coupling 632.8 nm light from a He–Ne laser. Our findings demonstrate the potential of fs-DLW as a novel approach for the fabrication of silk fibroin waveguides, which provides a versatile platform for optical and biomedical applications.

Facile Preparation of Mechanical Reinforced and Biocompatible Silk Gels

Nontoxic and controllable way to fabricate silk fibroin (SF) gel with high mechanical properties is of critical important to biomaterial in tissue engineering. Electrochemically triggered electrophoretic migration and electric charge of the silk molecules both contributed to SF microspheres and electronic gel (e-gel) formation by sol-gel transition. In this study, a novel silk pH e-gel with higher mechanical property was prepared by combining low-voltage electric fields with isoelectric point (pI) adjustment. This green process was mild and friendly without chemical crosslinker. Compressive modulus of the silk pH e-gel was up to 70 MPa that was significantly higher than that of SF gelation spontaneously. Furthermore, analysis of molecule conformation of the silk pH-e-gel demonstrated that most of random coil structures transformed into α-helix and a little β-sheet structures during this process. The silk pH e-gel was loaded with rhodamine B and showed an obvious sustainable release profile. Accumulation releasing amounts was approximately 60% at day 9. Cytocompatibility of the silk pH-e-gel was evaluated by epithelial cell. The results showed that the gels could support the cell growth and proliferation in vitro. Finally, gel biodegradation was assessed by protease XIV. After biodegradation for 28 days, remaining weight of the gel was about 20.23±2.59 wt%, indicating its good biodegradability. This novel process was established successfully by combining low voltage field with pH-control, which provided an alternative material for regenerative medicine.

Silk fibroin protein modified acellular dermal matrix for tissue repairing and regeneration

Worldwide the demand of skin-graft has been increasing day by day, for these different biomaterials and techniques have been used. In the present study, we have fabricated Silk Fibroin (SF) modified hybrid acellular goat-dermal matrix (SF-AGDM) by modifying the AGDM in different concentration SF― 5, 10, 15%, for enhancing the wound healing process.The grafts (AGDM and SF-AGDM) were evaluated for skin tissue regeneration by subjecting it through physical, chemical and biological characterization. SLS analysis showed the molecular weight of SF was 10,000 Da. Here, we found that SF-AGDM modified with low concentration of SF showed good porosity 78.56 ± 14.30% and pore size 74.69 ± 28.66 μm as similar to the AGDM. FTIR analysis showed the shifting of NH stretching (3400–3600 cm−1), amide I band at 3427 cm−1 and 1641 cm−1 and disappearance of the peaks of CH asymmetrical stretching (3000–2800 cm−1), amide II and amide III band, which indicate formation of amide linkage or other interaction between the SF protein and AGDM. In vitro cell culture studies by seeding 3 T3 mouse fibroblast cells on the scaffold revealed excellent cell viability, proliferation rate and adhesion in the scaffold. Pre-clinical study done in albino mice model showed within 14 days, all the wounds were completely cured, full thickness skin was regenerated without any significant inflammatory response. SF-ADGM results better healing as compared to the unmodified AGDM, which indicates the synergetic effect of SF coupled with acellular ECM based matrix.Thus, SF-AGDM is biocompatible, cost-effective material that can be potentially applied for tissue engineering application.

Electron beam irradiation effect on the mechanical and thermal properties of 2-D silk fibroin fabric/poly(lactic acid) biocomposites

Silk fibroin fabrics (SFF) were irradiated at various electron beam absorption doses. The irradiation effect on the breaking properties of SFF, the fiber topography, and the tensile, flexural and dynamic mechanical properties of 2-directional SFF/poly(lactic acid) (PLA) biocomposites were explored. The irradiation at 10kGy was most effective not only to increase the SFF breaking properties, but also to enhance the mechanical and thermal properties of SFF/PLA biocomposites. The microscopic analysis of fractured surfaces supported the irradiation dependence of the mechanical and thermal data, showing the increased interfacial bonding between the silk fiber and the PLA, particularly at 10kGy.

New fabrication method of silk fibroin plate and screw based on a centrifugal casting technique.

Recently, a newer generation of absorbable biomaterials has been developed from silk. Silk is approved by the US Food and Drug Administration, has robust mechanical features, and is biocompatible. Moreover, it offers the ability to be functionalized with bioactive compounds, making it ideal for use in new medical devices. Thus, many researchers have considered that absorbable devices made from silk may be able to overcome current limitations and could be used to meet a broader range of fixation needs. Here, we describe a novel method for the fabrication of silk fibroin (SF)-based bioabsorbable fixation systems using a centrifugal casting technique and incorporating a 3D printer. This approach allows us to create the desired geometric design for the fixation system easily. Moreover, our products demonstrated smoother surface profiles and more homogenous and dense cross-sectional architectures. Furthermore, our plates exhibited very similar mechanical properties compared with commercially used one, and our screws showed more than 70% of their initial mass after 7weeks on the enzymatic degradation test. On in vivo analysis, we found that our devices were well-maintained in the location of initial fixation, and new bone formation was also observed around this. By these results, we suggest that the SF-based plate/screw prepared by our novel method might be used for the internal fixation of fracture sites.

Surface modification of nanofibrous matrices via layer-by-layer functionalized silk assembly for mitigating the foreign body reaction

The inherent hydrophobicity and large surface area of electrospun synthetic polymeric scaffolds often cause non-specific protein adsorption, thereby influencing macrophage functions and eventually leading to fibrosis at the tissue-scaffold interface. This work reports fabrication of silk fibroin (SF)-functionalized electrospun polycaprolactone (PCL) fibers by single-component layer-by-layer assembly and decorate the SF with heparin disaccharide (HD), resulting in the non-covalent binding of interleukin-4 (IL-4) with the capacity to modulate macrophage polarization. A modified SF derivative was obtained by diazonium coupling and then covalently bonded with HD via click chemistry to eventually bind IL-4 efficiently and maintain its bioactivity. In vitro studies showed that IL-4 surface-functionalized nanofibrous scaffolds promoted polarization to M2 macrophages in the short-term. Importantly, in a murine subcutaneous implantation model, we found that promoting transient shifts in macrophage polarization at early stage can significantly inhibit the extent of the late foreign body reactions. Furthermore, the results of a transcriptomic profiling showed that MARK, PI3K, JNK and NF-κB signaling pathways played an important role in regulating the macrophage phenotypes in the SF/HD/IL-4-functionalized fibers. Our results suggest that such a strategy offers a new approach for utilizing biological agent surface functionalization to modulate the foreign body reaction to nanofibrous scaffolds.

Fabrication of Novel Silk Fibroin - LDHs Composite Arhitectures for Potential Bone Tissue Engineering

Nanocomposite materials have attracted a high interest for biomedical applications because their special properties related with structure and composition. In this paper we synthesized novel hydrogel nanocomposite materials special designed for hard tissue engineering. The nanocomposite materials are able to promote hydroxyapatite formation by alternating soaking mineralization demanded for increasing of cells biocompatibility and adhesion.

Silk fibroin-magnetic hybrid composite electrospun fibers for tissue engineering applications

Abstract This manuscript reports on the fabrication of silk fibroin (SF)-based magnetic electrospun fiber composites as scaffolds for tissue engineering applications. The magnetic responsiveness of the SF composite fibers was achieved by the inclusion of cobalt ferrite (CoFe2O4) or magnetite (Fe3O4) nanoparticles prior to processing the fibers via electrospinning. The influence of the processing parameters, including type and amount of nanoparticles in the composite, on the mean fiber size and size distribution was studied. Whereas the average diameter of pristine SF fibers was of 294 ± 53 nm, the inclusion of 5% of CoFe2O4 and Fe3O4 nanoparticles led to a slight increase in the fiber diameter. Nevertheless, the fiber diameter decreased with the higher nanoparticles loading. Regarding the physico-chemical properties of the fibrous mats, it was observed that the degree of crystallinity dropped from 67% of the pristine SF mats to 37% for the SF composites. On the other hand, the onset degradation temperature of the SF electrospun was not significantly altered by inclusion of ferrite nanoparticles. It is shown that the magnetization saturation increased with the nanoparticle filler content for both compositions (CoFe2O4/SF and Fe3O4/SF). Neither the SF pristine fibers nor the SF composites were cytotoxic, indicating their suitability for tissue engineering applications.

Preparation and Characterization of Silk Fibroin Based Antibacterial Biotextiles as Wound Dressing

A novel silk fibroin (SF) based bi-layered wound dressing was successfully developed by spraying chitosan (CS) solution on the ring spun silk fibroin fabric. SEM micrographs showed that chitosan solution formed a thin film on the degummed silk fibroin fabric, which had a connective porous structure. The antibacterial property, indirect cytotoxicity and the wound healing properties of the materials were investigated. Comparing with the commercial wound dressing, the bi-layered SF/CS wound dressing could accelerate the wound healing, and may be a potential choice for skin regeneration.

Reloadable Silk-Hydrogel Hybrid Scaffolds for Sustained and Targeted Delivery of Molecules.

Tunable repeated drug administration is often inevitable in a number of pathological cases. Reloadable 3D matrices for sustained drug delivery are predicted as a prospective avenue to realize this objective. This study was directed toward sonication-induced fabrication of novel reloadable Bombyx mori silk fibroin (SF) (4, 6, and 8 wt %) hydrogel, injected within 3D porous (8 wt %) scaffolds. The focus was to develop a dual-barrier reloadable depot system for sustained molecular cargo release. Both the varying SF concentration (4, 6, and 8 wt %) and the sonication time (30, 45, and 60 s) dictated the extent of cross-linking, β-sheet content, and porosity (1-10 μm) influencing the release behavior of model molecules. Release studies of model molecules (trypan blue, TB, 961 Da and bovine serum albumin, BSA, 66 kDa) for 28 days attested that the variations in their molecular weight, the matrix cross-linking density, and the scaffold-hydrogel interactions dictated the release behavior. The Ritger and Peppas equation was further fitted into the release behavior of model molecules from various SF matrices. The hybrid constructs exhibited high compressive strength along with in vitro compatibility using primary porcine chondrocytes and tunable enzymatic degradation as assessed for 28 days. The aptness of the constructs was evinced as a reloadable model molecule (BSA and fluorescein isothiocyanate-inulin, 3.9 kDa) depot system through UV-visible and fluorescence spectroscopic analyses. The novel affordable platform developed using silk scaffold-hydrogel hybrid constructs could serve as a sustained and reloadable drug depot system for administration of multiple and repeated drugs.

Fabrication of silk fibroin/eggshell nanofiber membranes for facemasks

We report our study on fabrication of soluble eggshell membrane (SESM) and silk fibroin (SF) nanofibers composite (SF/SESM) for facemasks by electrospinning. Biocompatibility of the SF and SESM, determined from hydrophilicity results, is exploited in SF/SESM nanocomposite for facemask application. The SF/SESM nanocomposites were prepared in different ratios of SF and SESM. The samples were characterized by scanning electron microscopy (SEM), FTIR and water droplet adsorption tests conducted via water contact angle (WCA) and water droplet diffusion. The results revealed that addition of SESM has insignificant effect on the electrospinnability of SF nanofibers in the studied ratios. The SEM results depicted regular morphology of the nanofibers except increase in nanofiber diameter with addition of SESM. The FTIR results confirmed respective peaks of SF and SESM in SF/SESM nanocomposite. WCA of the nanofibers decreased with addition of SESM such that for SF/SESM30, 30 % SESM, it reduced to 0 ° from 101 ° for pure SF nanofibers. The research results demonstrate SF/SESM30 nanocomposite as optimum ratio of SF and SESM for facemasks and other biomedical applications.

Fabrication of silk fibroin based three dimensional scaffolds for tissue engineering

Herein we report successful synthesis of silk fibroin (SF) three dimensional scaffolds (SF 3D-scaffold) from SF sponge and SF nanofibers. Both the nanofibers and sponge were prepared from Bombyx mori fibroin. The SF 3D-scaffold was prepared by electrospinning the fibroin nanofibers over the sponge. Surface morphology was determined by scanning electron microscopy (SEM), while nanofiber diameter and pore size were measured using imageJ software. Effect of spinning time on the pore size and cell adhesion was determined. Average diameter of the SF nanofibers was measured to be 320 nm and pore size was found to reduce with increasing spinning time, such that, for 1 h spinning time pore size was 231 µm and the same for 3.5 h was 4.1 µm. However, the number of pores increased with spinning time. The results confirmed adhesion of MC3T3-E1 cells on the SF sponge, SF nanofibers and SF three dimensional scaffolds. Higher cell adhesion was found on the three dimensional scaffold in comparison to the nanofibers and sponge, possibly due to highly porous structure with very small and numerous pores in the resultant composite; hence more cell adhesion sites. The cell adhesion result confirmed biocompatibility of the SF 3D-scaffold and hence its suitability for applications in tissue engineering.

Trilayered sulfated silk fibroin vascular grafts enhanced with braided silk tube

The scaffold component is a major barrier to the development of a clinically useful small-diameter tissue-engineered vascular graft. Scaffold requirements include matching the mechanical and structural properties with those of native vessels and optimizing the microenvironment for cell integration, adhesion, and growth. Trilayered sulfated silk fibroin graft was developed to mimic native tissue structure and function. Physical properties and cell studies were assessed to evaluate the viability of their usage in small-diameter tissue-engineered vascular grafts. Compared with previously fabricated silk fibroin vascular grafts, these trilayered grafts provided comparable water permeability, tensile strength, burst pressure, as well as suture retention strength, to saphenous veins for vascular grafts. In addition, the in vitro results showed good cytocompatibility of the trilayered grafts. These physical and cellular outcomes indicate potential utility of these trilayered sulfated silk fibroin grafts for small-diameter vascular grafts.

Fabrication of nanofibrous silkworm gland three-dimensional scaffold containing micro/nanoscale pores and study of its effects on adipose tissue-derived stem cell growth

An ideal tissue engineering scaffold requires a structure similar to that of the natural extracellular matrix. The structure of a scaffold is the most important parameter for determining the behavior and functions of cells. Therefore, the importance of designing a suitable scaffold has been greatly emphasized. Electrospinning has been extensively used to fabricate scaffolds for tissue engineering. In this study, we developed a nanofibrous scaffold made from silkworm gland (SG), which contains the silk protein fibroin and other proteins that have functions including antigenotoxicity and the promotion of cell growth. To investigate the physical and biological functions of SG nanofibrous (SGN) scaffold, we fabricated silk fibroin nanofibrous (SFN) scaffold as a control lacking the SG functional proteins. Field emission scanning electron microscope images revealed that the SGN three-dimensional scaffold had a homogenous pore distribution with a high porosity and interconnected pore walls. In biological tests, the SGN scaffold supported excellent cell viability and had a stronger antioxidant property compared to the SFN scaffold. In addition, the SGN scaffold efficiently supported the growth of adipose tissue-derived stem cells and their osteogenic and fibrocartilage differentiation. Therefore, this novel SGN three-dimensional scaffold shows high promise for use in tissue engineering.

Multi-biofunction of antimicrobial peptide-immobilized silk fibroin nanofiber membrane: Implications for wound healing

An antimicrobial peptide motif (Cys-KR12) originating from human cathelicidin peptide (LL37) was immobilized onto electrospun SF nanofiber membranes using EDC/NHS and thiol-maleimide click chemistry to confer the various bioactivities of LL37 onto the membrane for wound care purposes. Surface characterizations revealed that the immobilization density of Cys-KR12 on SF nanofibers could be precisely controlled with a high reaction yield. The Cys-KR12-immobilized SF nanofiber membrane exhibited antimicrobial activity against four pathogenic bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa) without biofilm formation on the membrane surface. It also facilitated the proliferation of keratinocytes and fibroblasts and promoted the differentiation of keratinocytes with enhanced cell-cell attachment. In addition, immobilized Cys-KR12 significantly suppressed the LPS-induced TNF-α expression of monocytes (Raw264.7) cultured on the membrane. These results suggest that a Cys-KR12-immobilized SF nanofiber membrane, which has multiple biological activities, would be a promising candidate as a wound dressing material. Statement of SignificanceThis research article reports various bioactivities of an antimicrobial peptide on electrospun silk fibroin nanofiber membrane. Recently, human cathelicidin peptide LL37 has been extensively explored as an alternative antibiotic material. It has not only a great antimicrobial activity but also a wide variety of bioactivities which can facilitate wound healing process. Especially, many studies on immobilization of LL37 or its analogues have shown the immobilization technique could improve performance of wound dressing materials or tissue culture matrices. Nevertheless, so far studies have only focused on the bactericidal effect of immobilized peptide on material surface. On the other hand, we tried to evaluate multi-biofunction of immobilized antimicrobial peptide Cys-KR12, which is the shortest peptide motif as an analogue of LL37. We fabricated silk fibroin nanofiber membrane as a model wound dressing by electrospinning and immobilized the antimicrobial peptide. As a result, we confirmed that the immobilized peptide can play multi-role in wound healing process, such as antimicrobial activity, facilitation of cell proliferation and keratinocyte differentiation, and inhibition of inflammatory cytokine expression. These findings have not been reported and can give an inspiration in wound-care application.