Siran Wang

Silk-tropoelastin protein films for nerve guidance

Peripheral nerve regeneration may be enhanced through the use of biodegradable thin film biomaterials as highly tuned inner nerve conduit liners. Dorsal root ganglion neuron and Schwann cell responses were studied on protein films comprising silk fibroin blended with recombinant human tropoelastin protein. Tropoelastin significantly improved neurite extension and enhanced Schwann cell process length and cell area, while the silk provided a robust biomaterial template. Silk-tropoelastin blends afforded a 2.4-fold increase in neurite extension, when compared to silk films coated with poly-d-lysine. When patterned by drying on grooved polydimethylsiloxane (3.5 μm groove width, 0.5 μm groove depth), these protein blends induced both neurite and Schwann cell process alignment. Neurons were functional as assessed using patch-clamping, and displayed action potentials similar to those cultured on poly(lysine)-coated glass. Taken together, silk-tropoelastin films offer useful biomaterial interfacial platforms for nerve cell control, which can be considered for neurite guidance, disease models for neuropathies and surgical peripheral nerve repairs.

Electrodeposited gels prepared from protein alloys

AIM Silk-tropoelastin alloys, composed of recombinant human tropoelastin and regenerated Bombyx mori silk fibroin, are an emerging, versatile class of biomaterials endowed with tunable combinations of physical and biological properties. Electrodeposition of these alloys provides a programmable means to assemble functional gels with both spatial and temporal controllability. MATERIALS & METHODS Tropoelastin-modified silk was prepared by enzymatic coupling between tyrosine residues. Hydrogel coatings were electrodeposited using two wire electrodes. RESULTS & DISCUSSION Mechanical characterization and in vitro cell culture revealed enhanced adhesive capability and cellular response of these alloy gels as compared with electrogelled silk alone. CONCLUSION These electro-depositable silk-tropoelastin alloys constitute a suitable coating material for nanoparticle-based drug carriers and offer a novel opportunity for on-demand encapsulation/release of nanomedicine.

Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk-based scaffolds.

Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases.

Degradation of silk films in multipocket corneal stromal rabbit models

Introduction The need for human cornea tissues continues to grow as an alternative option to donor tissues. Silk protein has been successfully used as a substrate to engineer corneal epithelium and stroma in vitro. Herein, we investigated the in vivo response and the effect of silk crystalline structure (beta sheet) on degradation rate of silk films in rabbit multipocket corneal models. Methods Three different surgical techniques (peripheral-median P-M, central-superficial C-S, central-deep C-D) were used to assess the in vivo response as well as the degradation profile of silk films with low, medium and high beta sheet (crystalline) content at 2 and 3 months after surgery. Results Approach C-D showed signs of sample degradation without inflammation, with one single incision and a pocket created by flushing air two thirds deep in the corneal stroma. In comparison, approaches P-M and C-S with multiple incisions presented manually dissected surgical pockets resulted in inflammation and possible extrusion of the samples, respectively. Low beta sheet samples lost structural integrity at 2 months after surgery C-D, while medium and high beta sheet content films showed initial evidence of degradation. Conclusions The in vivo response to the silk films was dependent on the location of the implant and pocket depth. Crystallinity content in silk films played a significant role in the timing of material degradation, without signs of inflammation and vascularization or changes in stromal organization.

Artificial Polymeric Scaffolds as Extracellular Matrix Substitutes for Autologous Conjunctival Goblet Cell Expansion

Purpose We fabricated and investigated polymeric scaffolds that can substitute for the conjunctival extracellular matrix to provide a substrate for autologous expansion of human conjunctival goblet cells in culture. Methods We fabricated two hydrogels and two silk films: (1) recombinant human collagen (RHC) hydrogel, (2) recombinant human collagen 2-methacryloylxyethyl phosphorylcholine (RHC-MPC) hydrogel, (3) arginine-glycine-aspartic acid (RGD) modified silk, and (4) poly-D-lysine (PDL) coated silk, and four electrospun scaffolds: (1) collagen, (2) poly(acrylic acid) (PAA), (3) poly(caprolactone) (PCL), and (4) poly(vinyl alcohol) (PVA). Coverslips and polyethylene terephthalate (PET) were used for comparison. Human conjunctival explants were cultured on scaffolds for 9 to 15 days. Cell viability, outgrowth area, and the percentage of cells expressing markers for stratified squamous epithelial cells (cytokeratin 4) and goblet cells (cytokeratin 7) were determined. Results Most of cells grown on all scaffolds were viable except for PCL in which only 3.6 ± 2.2% of the cells were viable. No cells attached to PVA scaffold. The outgrowth was greatest on PDL-silk and PET. Outgrowth was smallest on PCL. All cells were CK7-positive on RHC-MPC while 84.7 ± 6.9% of cells expressed CK7 on PDL-silk. For PCL, 87.10 ± 3.17% of cells were CK7-positive compared to PET where 67.10 ± 12.08% of cells were CK7-positive cells. Conclusions Biopolymer substrates in the form of hydrogels and silk films provided for better adherence, proliferation, and differentiation than the electrospun scaffolds and could be used for conjunctival goblet cell expansion for eventual transplantation once undifferentiated and stratified squamous cells are included. Useful polymer scaffold design characteristics have emerged from this study.

2.12 Silk Biomaterials

Natural biopolymers have applications in a variety of applications, including sensing, bioengineering, drug delivery, and tissue regeneration. In this review, we highlight the recent advances and application developments using the natural biopolymer, silk fibroin, derived from the cocoons of the Bombyx mori silkworm. The work highlighted here showcases the versatility of B. mori silk fibers for generation of a wide variety of material formats, including porous sponges, thin films, tubular architectures, fibers, hydrogels, and microparticles. Emphasis is placed on the role of the silk in each system, demonstrating methods for generation of long-term in vitro culture, implantable materials, or printable inks.