Silk Fibroin Biomaterials Research Articles

A Biomimetic Approach toward Enhancing Angiogenesis: Recombinantly Expressed Domain V of Human Perlecan Is a Bioactive Molecule That Promotes Angiogenesis and Vascularization of Implanted Biomaterials.

Angiogenic therapy involving delivery of pro‐angiogenic growth factors to stimulate new blood vessel formation in ischemic disease is promising but has seen limited clinical success due to issues associated with the need to deliver supra‐physiological growth factor concentrations. Bio‐inspired growth factor delivery utilizing the native growth factor signaling roles of the extracellular matrix proteoglycans has the potential to overcome many of the drawbacks of angiogenic therapy. In this study, the potential of the recombinantly expressed domain V (rDV) of human perlecan is investigated as a means of promoting growth factor signaling toward enhanced angiogenesis and vascularization of implanted biomaterials. rDV is found to promote angiogenesis in established in vitro and in vivo angiogenesis assays by potentiating endogenous growth factor signaling via its glycosaminoglycan chains. Further, rDV is found to potentiate fibroblast growth factor 2 (FGF2) signaling at low concentrations that in the absence of rDV are not biologically active. Finally, rDV immobilized on 3D porous silk fibroin biomaterials promotes enhanced vascular ingrowth and integration of the implanted scaffolds with the surrounding tissue. Together, these studies demonstrate the important role of this biologically active perlecan fragment and its potential in the treatment of ischemia in both native and bioengineered tissues.

Higher Gene Expression Related to Wound Healing by Fibroblasts on Silk Fibroin Biomaterial than on Collagen.

Silk fibroin (SF), which offers the benefits of biosafety, biocompatibility, and mechanical strength, has potential for use as a good biomedical material, especially in the tissue engineering field. This study investigated the use of SF biomaterials as a wound dressing compared to commercially available collagen materials. After human fibroblasts (WI-38) were cultured on both films and sponges, their cell motilities and gene expressions related to wound repair and tissue reconstruction were evaluated. Compared to the collagen film (Col film), the SF film induced higher cell motility; higher expressions of genes were observed on the SF film. Extracellular matrix production-related genes were up-regulated in WI-38 fibroblasts cultured on the SF sponges. These results suggest that SF-based biomaterials can accelerate wound healing and tissue reconstruction. They can be useful biomaterials for functional wound dressings.

Impact of the magnetic field on 3T3-E1 preosteoblasts inside SMART silk fibroin-based scaffolds decorated with magnetic nanoparticles.

This paper reports the impact of the magnetic field on 3T3-E1 preosteoblasts within silk-fibroin scaffolds decorated with magnetic nanoparticles. Scaffolds were prepared from silk fibroin and poly(2-hydroxyethyl methacrylate) template in which magnetite nanoparticles were embedded. The presence of the magnetite specific peaks within scaffolds compositions was evidenced by RAMAN analysis. Structural investigation was done by XRD analysis and morphological information including internal structure was obtained through SEM analysis. Geometrical evaluation (size and shape), crystalline structure of magnetic nanoparticles and the morphology of the silk fibroin scaffolds were investigated by HR-TEM. Magnetic nanoparticles were distributed within scaffolds structure. Biomineralization of hydroxyapatite on silk fibroin scaffolds with and without magnetic nanoparticles was investigated by an alternate soaking process. SEM images showed that the magnetic scaffolds were covered in an almost continuously film, which has a phase with nanostructured characteristics. This phase, which has as main components Ca and P, is made of lamellar formations. The design of an original magnetic 3D cell culture setup allowed us to observe cellular modifications under the exposure to magnetic field in the presence of magnetic silk fibroin biomaterials. The cellular proliferation potential of 3T3-E1 cell line was found increased under the magnetic field, especially in the presence of the magnetite nanoparticles. In addition, we showed that the low static magnetic field positively impacts on the osteogenic differentiation potential of the cells inside the biomimetic magnetic scaffolds.

Flexible Water-Absorbing Silk-Fibroin Biomaterial Sponges with Unique Pore Structure for Tissue Engineering.

Silk fibroin (SF) scaffolds are widely used in tissue engineering due to their biocompatibility and slow biodegradability. However, the relatively stiff mechanical properties and low permeability of these systems can limit some applications. In this study, a new type of water-stable silk sponge (ASF-PEG-S) was obtained by inducing nanoparticle (50-300 nm in diameter) formation in SF solution by autoclaving followed by freeze-drying and rinsing the dry sponges with low-molecular weight (400 Da) polyethylene glycol (PEG400) to induce SF β-sheet structure formation and thus stability in water. With further extraction, the SF nanoparticles embedded in the sponges were removed, leaving nanopores in the walls of round-shaped micro-size pores. The unique pore structure resulted in enhanced permeability and flexibility of ASF-PEG-S when compared to other types of SF sponges, especially with respect to commonly used methanol-annealed SF sponges. In addition, ASF-PEG-S absorbed water nearly 40 times more than its dry weight, while the methanol-annealed sponges absorbed half this amount. When human fibroblasts were seeded and cultured on ASF-PEG-S versus traditional SF methanol-processed sponges, improved cell encapsulation, distribution, and consistency in growth were observed, suggesting utility in tissue engineering and tissue repair applications in the future.

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 β-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.

Effect of Degumming Methods on the Degradation Behavior of Silk Fibroin Biomaterials

Degradation behavior is a key consideration in the field of silk fibroin (SF) biomaterials. Degumming to remove sericin is a prerequisite for SF purification; however, the impact of degumming on the degradation behavior of SF biomaterials has not been established. In this study, two different degumming systems, Na2CO3 and NaHCO3, were used. Na2CO3 exhibited higher degumming efficiency but caused greater degradation of the fibroin. The results demonstrated that NaHCO3 degumming could afford regenerated SF with higher molecular weight, resulting in SF films with higher mechanical strengths. The enzymatic degradation behaviors indicated that the SF films prepared by the Na2CO3 degumming process showed faster degradation, revealing that the choice of degumming method has a substantial impact on the biodegradation of SF-based materials. The results showed that manipulating the degumming conditions can be used to tune the molecular weight of the SF, in turn providing control over the degradation rate of SF biomaterials.

Silk-Based Biomaterials in Cutaneous Wound Healing: A Systematic Review.

Effective wound dressings should promote healing through cellular migration, neovascularization, and re-epithelialization. Silk fibroin (SF) and silk sericin (SS) are reported to have very good biocompatibility, excellent mechanical properties, and controlled biodegradability. This review investigates the use and performance of silk-based biomaterials in cutaneous wounds within in vitro, in vivo, and randomized controlled studies. Study authors conducted a comprehensive literature search on the use of silk-based dressings in cutaneous wound healing and investigated reports of the advantages and disadvantages of SF and SS along with these materials' methods of characterization, cell migration, neovascularization, wound closure, and cytotoxicity. In vitro and in vivo animal models have shown SF-based biomaterials promote good cellular adhesion and fibroblast proliferation in cutaneous wounds. The porosity and silk concentration of silk-based scaffolds are key determinants of biodegradation and plasmatic imbibition capabilities and can help promote wound healing. In reviewed studies, SF biomaterials promoted neovascularization as early as 7 days and better than common dressings, demonstrating low cytotoxicity and immunogenicity. That said, a concern with the use of SS is the tendency to cause a hypersensitivity reaction. The benefits of silk-based biomaterials seem evident based on promising preclinical studies. Both SF and SS have been shown to have excellent wound healing properties by promoting cell attachment, migration, and collagen deposition. The authors encourage the use of SF and SS in more trials for wound healing.

Avidin Adsorption to Silk Fibroin Films as a Facile Method for Functionalization

Silk fibroin biomaterials are highly versatile in terms of materials formation and functionalization, with applications in tissue engineering and drug delivery, but necessitate modifications for optimized biological activity. Herein, a facile, avidin-based technique is developed to noncovalently functionalize silk materials with bioactive molecules. The ability to adsorb avidin to silk surfaces and subsequently couple biotinylated macromolecules via avidin-biotin interaction is described. This method better preserved functionality than standard covalent coupling techniques using carbodiimide cross-linking chemistry. The controlled release of avidin from the silk surface was demonstrated by altering the adsorption parameters. Application of this technique to culturing human foreskin fibroblasts (hFFs) and human mesenchymal stem cells (hMSCs) on arginine-glycine-aspartic-acid-modified (RGD-modified) silk showed increased cell growth over a seven-day period. This technique provides a facile method for the versatile functionalization of silk materials for biomedical applications including tissue engineering, drug delivery, and biological sensing.

Plasma Ion Implantation of Silk Biomaterials Enabling Direct Covalent Immobilization of Bioactive Agents for Enhanced Cellular Responses.

Silk fibroin isolated from Bombyx mori cocoons is a promising material for a range of biomedical applications, but it has no inherent cell-interactive domains, necessitating functionalization with bioactive molecules. Here we demonstrate significantly enhanced cell interactions with silk fibroin biomaterials in the absence of biofunctionalization following surface modification using plasma immersion ion implantation (PIII). Further, PIII treated silk fibroin biomaterials supported direct covalent immobilization of proteins on the material surface in the absence of chemical cross-linkers. Surface analysis after nitrogen plasma and PIII treatment at 20 kV revealed that the silk macromolecules are significantly fragmented, and at the higher fluences of implanted ions, surface carbonization was observed to depths corresponding to that of the ion penetration. Consistent with the activity of radicals created in the treated surface layer, oxidation was observed on contact with atmospheric oxygen and the PIII treated surfaces were capable of direct covalent immobilization of bioactive macromolecules. Changes in thickness, amide and nitrile groups, refractive index, and extinction coefficient in the wavelength range 400-1000 nm as a function of ion fluence are presented. Reactions responsible for the restructuring of the silk surface under ion beam treatment that facilitate covalent binding of proteins and a significant improvement in cell interactions on the modified surface are proposed.

Biomimetic Silk Scaffolds with an Amorphous Structure for Soft Tissue Engineering.

Fine tuning physical cues of silk fibroin (SF) biomaterials to match specific requirements for different soft tissues would be advantageous. Here, amorphous SF nanofibers were used to fabricate scaffolds with better hierarchical extracellular matrix (ECM) mimetic microstructures than previous silk scaffolds. Kinetic control was introduced into the scaffold forming process, resulting in the direct production of water-stable scaffolds with tunable secondary structures and thus mechanical properties. These biomaterials remained with amorphous structures, offering softer properties than prior scaffolds. The fine mechanical tunability of these systems provides a feasible way to optimize physical cues for improved cell proliferation and enhanced neovascularization in vivo. Multiple physical cues, such as partly ECM mimetic structures and optimized stiffness, provided suitable microenvironments for tissue ingrowth, suggesting the possibility of actively designing bioactive SF biomaterials. These systems suggest a promising strategy to develop novel SF biomaterials for soft tissue repair and regenerative medicine.

The Wonders of Silk Fibroin Biomaterials in the Treatment of Breast Cancer.

Breast cancer has continued to be a cause of increasing morbidity and mortality in women, being the most common cause of cancer-related deaths among them. Its management using chemotherapy is continually plagued with problems of systemic toxicity, lack of compliance by patients, and inadequate targeting of cancer cells. The future of breast cancer chemotherapy will likely involve the use of biocompatible high cell-targeting capacity drug-delivery vehicles like silk fibroin to ameliorate these problems. The utilization of silk fibroin nanoparticles to deliver cytotoxic drugs provides specificity, optimal entrapment, improved therapeutic index, and maximal breast cancer cell toxicity with minimal or no collateral damage to surrounding normal cells. The silk fibroin obtained from the cocoon of the Bombyx morii worm is processed and degummed to remove the sericin component; it is then made into nanoparticles utilizing the desolvation, ionic gelation, or electrospray method. It is then loaded with an appropriate chemotherapeutic drug (e.g., carboplatin), and characterization is performed using physico-chemical methods such as fourier-transform infrared spectroscopy, dynamic light scattering, or transmission electron microscopy. The nanoparticles are then tested for cytoxicity, and the induction of apoptosis on breast cancer cell lines MC-7 (Her2-) and MDA-MB-453 (Her2+). Toxicity and apoptosis are assessed using the MTT assay and ELISA methods, respectively. Silk fibroin has been demonstrated in various studies to be a very useful tool in specific active or passive drug delivery to target cancer cells, thus ensuring a maximum destruction and minimum damage to normal surrounding cells, which decreases systemic toxicity and enhances drug efficacy.

Fractal characteristics of the microvascular network: A useful index to assess vascularization level of porous silk fibroin biomaterial.

The neovascularization of biomaterials for tissue engineering is not only related to growth of capillaries but also involves appropriate hierarchy distribution of the microvessels. In this study, we proposed hierarchy distribution contrast method which can assess vascular transport capacity, in order to examine the hierarchy distribution of the neovessels during vascularization of the porous silk fibroin biomaterials implanted into rats and its evolution. The results showed that the fractal characteristics appeared toward the end of the vascularization stages, and the structure of the microvascular network after 3weeks of implantation was similar to the fractal microvascular tree with bifurcation exponent x=3 and fractal dimension D=1.46, which became a sign of maturation of the regenerative vasculature. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2276-2290, 2017.

Wound Healing: Silk Fibroin Biomaterial Shows Safe and Effective Wound Healing in Animal Models and a Randomized Controlled Clinical Trial (Adv. Healthcare Mater. 10/2017)

Silk fibroin is a natural protein derived from Bombyx mori cocoons, and shows potential in tissue repair applications due to its excellent biomedical properties. The silk fibroin film, developed by Xiaohui Zou, Hongwei Ouyang, and co-workers from Zhejiang University in article number 1700121, is a highly transparent, biocompatible, air-permeable, waterproof and bacteria barrier scaffold. It significantly reduces the time to wound healing and incidence of adverse events compared to commercial dressing.

Silk Fibroin Biomaterial Shows Safe and Effective Wound Healing in Animal Models and a Randomized Controlled Clinical Trial.

Due to its excellent biological and mechanical properties, silk fibroin has been intensively explored for tissue engineering and regenerative medicine applications. However, lack of translational evidence has hampered its clinical application for tissue repair. Here a silk fibroin film is developed and its translational potential is investigated for skin repair by performing comprehensive preclinical and clinical studies to fully evaluate its safety and effectiveness. The silk fibroin film fabricated using all green chemistry approaches demonstrates remarkable characteristics, including transmittance, fluid handling capacity, moisture vapor permeability, waterproofness, bacterial barrier properties, and biocompatibility. In vivo rabbit full-thickness skin defect study shows that the silk fibroin film effectively reduces the average wound healing time with better skin regeneration compared with the commercial wound dressings. Subsequent assessment in porcine model confirms its long-term safety and effectiveness for full-thickness skin defects. Finally, a randomized single-blind parallel controlled clinical trial with 71 patients shows that the silk fibroin film significantly reduces the time to wound healing and incidence of adverse events compared to commercial dressing. Therefore, the study provides systematic preclinical and clinical evidence that the silk fibroin film promotes wound healing thereby establishing a foundation towards its application for skin repair and regeneration in the clinic.

A Review of Structure Construction of Silk Fibroin Biomaterials from Single Structures to Multi-Level Structures.

The biological performance of artificial biomaterials is closely related to their structure characteristics. Cell adhesion, migration, proliferation, and differentiation are all strongly affected by the different scale structures of biomaterials. Silk fibroin (SF), extracted mainly from silkworms, has become a popular biomaterial due to its excellent biocompatibility, exceptional mechanical properties, tunable degradation, ease of processing, and sufficient supply. As a material with excellent processability, SF can be processed into various forms with different structures, including particulate, fiber, film, and three-dimensional (3D) porous scaffolds. This review discusses and summarizes the various constructions of SF-based materials, from single structures to multi-level structures, and their applications. In combination with single structures, new techniques for creating special multi-level structures of SF-based materials, such as micropatterning and 3D-printing, are also briefly addressed.

Roles of Mesenchymal Stem Cells in Spinal Cord Injury.

Spinal cord injury (SCI) represents one of the most complicated and heterogeneous pathological processes of central nervous system (CNS) impairments, which is still beyond functional regeneration. Transplantation of mesenchymal stem cells (MSCs) has been shown to promote the repair of the injured spinal cord tissues in animal models, and therefore, there is much interest in the clinical use of these cells. However, many questions which are essential to improve the therapy effects remain unanswered. For instance, the functional roles and related molecular regulatory mechanisms of MSCs in vivo are not yet completely determined. It is important for transplanted cells to migrate into the injured tissue, to survive and undergo neural differentiation, or to play neural protection roles by various mechanisms after SCI. In this review, we will focus on some of the recent knowledge about the biological behavior and function of MSCs in SCI. Meanwhile, we highlight the function of biomaterials to direct the behavior of MSCs based on our series of work on silk fibroin biomaterials and attempt to emphasize combinational strategies such as tissue engineering for functional improvement of SCI.

Novel fabrication of fluorescent silk utilized in biotechnological and medical applications

Silk fibroin (SF) is a natural polymer widely used and studied for diverse applications in the biomedical field. Recently, genetically modified silks, particularly fluorescent SF fibers, were reported to have been produced from transgenic silkworms. However, they are currently limited to textile manufacturing. To expand the use of transgenic silkworms for biomedical applications, a solution form of fluorescent SF needed to be developed. Here, we describe a novel method of preparing a fluorescent SF solution and demonstrate long-term fluorescent function up to one year after subcutaneous insertion. We also show that fluorescent SF labeled p53 antibodies clearly identify HeLa cells, indicating the applicability of fluorescent SF to cancer detection and bio-imaging. Furthermore, we demonstrate the intraoperative use of fluorescent SF in an animal model to detect a small esophageal perforation (0.5 mm). This study suggests how fluorescent SF biomaterials can be applied in biotechnology and clinical medicine.

Cell-debris self-immobilized thermophilic esterase: A novel biocatalyst for synthesizing aliphatic polyesters

References [1] E.M. Pritchard, D.L. Kaplan, Silk fibroin biomaterials for controlled release drug delivery, Exp. Opin. Drug. Deliv. 8 (2001) 797–811. [2] L.P. Yan, J.M. Oliveira, A.L. Oliveira, S.G. Caridade, J.F. Mano, R.L. Reis, Macro/ microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications, Acta Biomater. 8 (2012) 289–301. [3] S.J. Min, X. Gao, C.M. Han, Y. Chen, M.Y. Yang, L.J. Zhu, H.P. Zhang, L. Liu, J.M. Yao, Preparation of a silk fibroin spongy wound dressing and its therapeutic efficiency in skin defects, J. Biomater. Sci. 23 (2012) 97–110.

Encapsulation of oil in silk fibroin biomaterials

ABSTRACTMicroencapsulation is becoming increasingly important in the food, cosmetics, and medicinal industries due to its potential for stabilization and delivery of volatile and delicate compounds. Novel food‐safe techniques for encapsulating oil in silk biomaterials using emulsion‐based processes that exploit silk's unique properties (including amphiphilicity, biocompatibility, aqueous and ambient processing, and tunable physical crosslinking behavior) are described. The sonication‐induced self‐assembly of silk previously applied to hydrogel fabrication replaced the use of the thermal or chemical suspension crosslinking traditionally used to stabilize the aqueous protein phase in emulsions. Stable, physically crosslinked silk micro‐ and macro‐particles loaded with oil or water‐soluble dye were produced by aliquoting sonicated silk solutions into an oil bath. Oil micro‐droplets emulsified in aqueous silk solutions did not impede the self‐assembly of silk into films or hydrogel networks. In O/W/O emulsions, particle morphology and silk permeability to a model lipophilic dye in the interior phase were controllable via processing. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2014, 131, 39990.

Human Placenta-Derived Mesenchymal Stem Cells with Silk Fibroin Biomaterial in the Repair of Articular Cartilage Defects

Cartilage tissue engineering requires a porous biodegradable scaffold and nonimmunogenic cells with chondrogenic potential. In this study, the ability of the placenta-derived mesenchymal stem cells (PMSCs) to grow on silk fibroin (SF) biomaterial was determined, and the potential of a SF biomaterial serving as a delivery vehicle for human PMSCs in a rabbit articular cartilage defects model was evaluated. Human PMSCs were maintained in vitro in an allogeneic mixed lymphocyte reactions (MLR) system to investigate the suppressive effects on T cell proliferation. A total of 12 healthy adult New Zealand rabbits were implanted with a PMSC/SF biomaterial complex after articular cartilage defects of the femoral condyle in the knee were established. The repair of the articular cartilage defects was observed after 4 weeks, 8 weeks, and 12 weeks. Results from the MLR indicated that human PMSCs inhibited rabbit T cell responses. Knee damage was repaired by the newly formed hyaline cartilage, and within 12 weeks there was neither degeneration nor infiltration with lymphocytes or leukocytes, and no silk fibroin biomaterial residue was detected. In conclusion, the silk fibroin biomaterial can be applied as a new scaffold for cartilage tissue engineering, and implantation of human PMSCs on the cartilage can enhance repair of articular cartilage defects in a rabbit model.