Silk Protein Films Research Articles

Bombyx mori silk protein films microprocessing with a nanosecond ultraviolet laser and a femtosecond laser workstation: theory and experiments

Laser microprocessing of several biopolymers from renewable resources is studied. Three proteinic materials were either extracted from the extracellular matrix like Silk Fibroin/Sericin and collagen, or coming from a commercial source like gelatin. All can find future applications in biomedical experimentation, in particular for cell scaffolding. Films of ∼hundred of microns thick were made by aqueous solution drying and laser irradiation. Attention is paid to the properties making them processable with two laser sources: the ultraviolet and nanosecond (ns) KrF (248 nm) excimer and the infrared and femtosecond (fs) Yb:KGW laser. The UV radiation is absorbed in a one-photon resonant process to yield ablation and the surface foaming characteristics of a laser-induced pressure wave. To the contrary, resonant absorption of the IR photons of the fs laser is not possible and does not take place. However, the high field of the intense I>∼1012 W/cm2 femtosecond laser pulse ionizes the film by the multiphoton absorption followed by the electron impact mechanism, yielding a dense plasma capable to further absorb the incident radiation of the end of the pulse. The theoretical model of this absorption is described in detail, and used to discuss the presented experimental effects (cutting, ablation and foaming) of the fs laser. The ultraviolet laser was used to perform simultaneous multiple spots experiments in which energetic foaming yields melt ejection and filament spinning. Airborne nanosize filaments “horizontally suspended by both ends” (0.25 μm diameter and 10 μm length) of silk biopolymer were observed upon irradiation with large fluences.

The characterization of protein release from sericin film in the presence of an enzyme: Towards fibroblast growth factor-2 delivery

Aqueous preparations of silk protein (sericin) films were prepared to evaluate their biodegradation properties. In the absence of trypsin, sericin film swelled rapidly, kept its shape, and remained unaltered for 28 days or longer due to form β-sheet structures. In the presence of trypsin, sericin film gradually degraded; since the rate depended on the concentration of trypsin, the films likely underwent enzymatic hydrolysis. Sericin film incorporating the model protein drug fluorescein isothiocyanate–albumin (FA) also gradually degraded in the presence of trypsin and resulted in the sustained release of FA for 2 weeks or longer; in contrast, FA release was quite slow in the absence of trypsin. It is expected that sericin film has potential as a biodegradable and drug-releasing carrier. To evaluate the practical applicability of sericin film for the repair of defective tissues, fibroblast growth factor-2 (FGF-2) was incorporated into sericin films and the films were implanted on skull defects in rats. Whereas FGF-2 release was suppressed in the absence of trypsin in vitro, it appears that FGF-2, immobilized by ionic interactions between sericin and FGF-2, can be sustained-released in vivo from films incorporating 2500 or 250 ng of FGF-2 to support the growth of tissue around wounds.

Safety evaluation of silk protein film (A novel wound healing agent) in terms of acute dermal toxicity, acute dermal irritation and skin sensitization

Acute dermal toxicity study was conducted in rats. The parameters studied were body weight, serum biochemistry and gross pathology. The animals were also observed for clinical signs and mortality after the application of test film. The dermal irritation potential of silk protein film was examined using Draize test. In the initial test, three test patches were applied sequentially for 3 min, 1 and 4 hours, respectively, and skin reaction was graded. The irritant or negative response was confirmed using two additional animals, each with one patch, for an exposure period of 4 hours. The responses were scored at 1, 24, 48 and 72 hours after the patch removal. Skin sensitization study was conducted according to Buehler test in guinea pigs, in which on day 0, 7 and 14, the animals were exposed to test material for 6 hours (Induction phase) and on day 28, the animals were exposed for a period of 24 hours (Challenge phase). The skin was observed and recorded at 24 and 48 hours after the patch removal. In acute dermal toxicity study, the rats dermally treated with silk film did not show any abnormal clinical signs and the body weight, biochemical parameters and gross pathological observations were not significantly different from the control group. In acute dermal irritation study, the treated rabbits showed no signs of erythema, edema and eschar, and the scoring was given as “0” for all time points of observations according to Draize scoring system. In skin sensitization study, there were no skin reactions 24 and 48 hours after the removal of challenge patch, which was scored “0” based on Magnusson/Kligman grading scale.

Quantifying Osteogenic Cell Degradation of Silk Biomaterials

The degradation of silk protein films by human mesenchymal stem cells (hMSCs), osteoblasts and osteoclasts, cells involved in osteogenic functions in normal and diseased bone, was assessed in vitro. The involvement of specific matrix metalloproteinases (MMPs) and integrin signaling in the degradation process was determined. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to quantitatively compare degradation by the different cell types using surface patterned silk films. Osteoblasts and osteoclasts demonstrated significant degradation of the silk films in vitro in comparison to the hMSCs and the film controls without cells. The osteoclasts degraded the silk films the most and also generated the highest level of MMPs 1 and 2. The osteoblasts upregulated integrins α5 and β1, while the osteoclasts upregulated integrins α2 and β1. There was significant contrast in responses on the silk matrices between osteogenic cells versus undifferentiated hMSCs to illustrate in vitro the role of cell type on matrix remodeling. These are important issues in matching biomaterial matrix features and studies in vitro to remodeling in vivo, in both normal and disease tissue systems. Cell populations and niche factors impact tissue regeneration, wound healing, physiological state, and the ability to better understand the role of different cell types is critical to overall regenerative outcomes.

Silk film biomaterials for cornea tissue engineering

Biomaterials for corneal tissue engineering must demonstrate several critical features for potential utility in vivo, including transparency, mechanical integrity, biocompatibility and slow biodegradation. Silk film biomaterials were designed and characterized to meet these functional requirements. Silk protein films were used in a biomimetic approach to replicate corneal stromal tissue architecture. The films were 2 μm thick to emulate corneal collagen lamellae dimensions, and were surface patterned to guide cell alignment. To enhance trans-lamellar diffusion of nutrients and to promote cell–cell interaction, pores with 0.5–5.0 μm diameters were introduced into the silk films. Human and rabbit corneal fibroblast proliferation, alignment and corneal extracellular matrix expression on these films in both 2D and 3D cultures were demonstrated. The mechanical properties, optical clarity and surface patterned features of these films, combined with their ability to support corneal cell functions suggest that this new biomaterial system offers important potential benefits for corneal tissue regeneration.

Conformation transition in silk protein films monitored by time-resolved Fourier transform infrared spectroscopy: effect of potassium ions on Nephila spidroin films.

We used time-resolved Fourier transform infrared spectroscopy (FTIR) to follow a conformation transition in Nephila spidroin film from random coil and/or helical structures to beta-sheet induced by the addition of KCl from 0.01 to 1.0 mol/L in D(2)O. Time series difference spectra showed parallel increases in absorption at 1620 and 1691 cm(-)(1), indicating formation of beta-sheet, together with a coincident loss of intensity of approximately 1650 cm(-)(1), indicating decrease of random coil and/or helical structures. Increase in KCl concentration produced an increased rate of the conformation transition that may attributable to weakening of hydrogen bonds within spidroin macromolecules. The conformation transition was a biphasic process with [KCl] > or = 0.3 mol/L but monophasic with [KCl] < or = 0.1 mol/L. This suggests that, at high KCl concentrations, segments of the molecular chain are adjusted first and then the whole molecule undergoes rearrangement. We discuss the possible significance of these findings to an understanding of the way that spiders spin silk.

Laser Ablation of Silk Protein (Fibroin) Films

Fibroin is the main protein component of silk and is expected to have functional applications in bioelectronics and medicine. We investigated nanosecond (ns) pulsed laser ablation of solid fibroin films with/without a dye as a photosensitizer. Laser lights at 248 nm and 532/355/351 nm excited the peptide bond of fibroin and the dye, respectively. The neat film irradiated at 248 nm was scarcely accessible to etching and swelling, and instead, a microscopic pattern (structure) was formed. In contrast, for ablation of the doped film at 532/355/351 nm, we found marked swelling (height ∼500 µm) and deep etching (depth ∼10 µm) on the irradiated surfaces. The dye-photosensitized ablation was brought about by a photothermal mechanism, whereas ablation of neat films may be induced by another process, such as a photochemical one. The ablation processes are discussed in terms of the properties of fibroin and the mode of excitation.